Issue published August 1, 2003

H epatitis delta virus (HDV) is a cause of acute and chronic liver disease for which no effective therapy is currently available. Previous research has demonstrated that prenylation of the large HDV antigen is essential for viral assembly. A new report describes a novel small animal model for HDV replication and demonstrates that prenylation inhibitors are highly effective at clearing viremia and thus have potential relevance for the treatment of chronic delta hepatitis.

A lzheimer disease (AD) is characterized by the progressive accumulation of amyloid β protein (Aβ) in areas of the brain serving cognitive functions such as memory and language. The first of two separate reports (see the related articles beginning on pages 415 and 440) reveals that intrinsic T cell reactivity to the self-antigen Aβ exists in many humans and increases with age. This finding has implications for the design of Aβ vaccines. The second report demonstrates that a number of FDA- approved nonsteroidal anti-inflammatory drugs are capable of lowering Aβ levels in mice. The work suggests that further testing of the therapeutic utility of these types of compounds for the potential treatment of AD is warranted.

E ndocannabinoids, endogenous ligands of cannabinoid receptor type 1 (CB1), have emerged as novel and important regulators of energy homeostasis. A report in this issue demonstrates reduced body weight, fat mass, and appetite in CB1^–/– mice. Examination of the underlying mechanisms reveals a dual role for endocannabinoids as they affect both appetite and peripheral lipolysis.

A delicate balance exists between ECM synthesis and degradation such that interruption of the corresponding pathways results in increased plasminogen activator inhibitor-1 (PAI-1), pathological matrix accumulation, and glomerulosclerosis. A new study demonstrates that therapy with a mutant PAI-1 increases matrix turnover and reduces glomerulosclerosis by competing with endogenous PAI-1, suggesting therapeutic utility in the treatment of fibrotic renal disease.

I nflammation and airway remodeling are two responses readily apparent in asthma and other inflammatory disorders of the airway and lungs. Both adenosine and IL-13 play critical roles in contributing pathways. A new study reveals a previously unrecognized interaction between adenosine and IL-13 that indicates a mutual stimulation that may contribute to the nature and severity of airway inflammation and fibrosis.

I L-13 is an important mediator of inflammation and remodeling. We hypothesized that adenosine accumulation, alterations in adenosine receptors, and adenosine–IL-13 autoinduction are critical events in IL-13–induced pathologies. To test this, we characterized the effects of IL-13 overexpression on the levels of adenosine, adenosine deaminase (ADA) activity, and adenosine receptors in the murine lung. We also determined whether adenosine induced IL-13 in lungs from ADA-null mice. IL-13 induced an inflammatory and remodeling response that caused respiratory failure and death. During this response, IL-13 caused a progressive increase in adenosine accumulation, inhibited ADA activity and mRNA accumulation, and augmented the expression of the A₁, A_2B, and A₃ but not the A_2A adenosine receptors. ADA enzyme therapy diminished the IL-13–induced increase in adenosine, inhibited IL-13–induced inflammation, chemokine elaboration, fibrosis, and alveolar destruction, and prolonged the survival of IL-13–transgenic animals. In addition, IL-13 was strongly induced by adenosine in ADA-null mice. These findings demonstrate that adenosine and adenosine signaling contribute to and influence the severity of IL-13–induced tissue responses. They also demonstrate that IL-13 and adenosine stimulate one another in an amplification pathway that may contribute to the nature, severity, progression, and/or chronicity of IL-13 and/or Th2-mediated disorders.

H epatocyte nuclear factors-3 (Foxa-1–3) are winged forkhead transcription factors that regulate gene expression in the liver and pancreatic islets and are required for normal metabolism. Here we show that Foxa-2 is expressed in preadipocytes and induced de novo in adipocytes of genetic and diet-induced rodent models of obesity. In preadipocytes Foxa-2 inhibits adipocyte differentiation by activating transcription of the Pref-1 gene. Foxa-2 and Pref-1 expression can be enhanced in primary preadipocytes by growth hormone, suggesting that the antiadipogenic activity of growth hormone is mediated by Foxa-2. In differentiated adipocytes Foxa-2 expression leads to induction of gene expression involved in glucose and fat metabolism, including glucose transporter-4, hexokinase-2, muscle-pyruvate kinase, hormone-sensitive lipase, and uncoupling proteins-2 and -3. Diet-induced obese mice with haploinsufficiency in Foxa-2 (Foxa-2^+/–) develop increased adiposity compared with wild-type littermates as a result of decreased energy expenditure. Furthermore, adipocytes of these Foxa-2^+/– mice exhibit defects in glucose uptake and metabolism. These data suggest that Foxa-2 plays an important role as a physiological regulator of adipocyte differentiation and metabolism.

E ctopic calcification is a frequent complication of many degenerative diseases. Here we identify the serum protein α₂–Heremans-Schmid glycoprotein (Ahsg, also known as fetuin-A) as an important inhibitor of ectopic calcification acting on the systemic level. Ahsg-deficient mice are phenotypically normal, but develop severe calcification of various organs on a mineral and vitamin D–rich diet and on a normal diet when the deficiency is combined with a DBA/2 genetic background. This phenotype is not associated with apparent changes in calcium and phosphate homeostasis, but with a decreased inhibitory activity of the Ahsg-deficient extracellular fluid on mineral formation. The same underlying principle may contribute to many calcifying disorders including calciphylaxis, a syndrome of severe systemic calcification in patients with chronic renal failure. Taken together, our data demonstrate a critical role of Ahsg as an inhibitor of unwanted mineralization and provide a novel therapeutic concept to prevent ectopic calcification accompanying various diseases.

H epatic lipase (HL) has a well-established role in lipoprotein metabolism. However, its role in atherosclerosis is poorly understood. Here we demonstrate that HL deficiency raises the proatherogenic apoB-containing lipoprotein levels in plasma but reduces atherosclerosis in lecithin cholesterol acyltransferase (LCAT) transgenic (Tg) mice, similar to results previously observed with HL-deficient apoE-KO mice. These findings suggest that HL has functions that modify atherogenic risk that are separate from its role in lipoprotein metabolism. We used bone marrow transplantation (BMT) to generate apoE-KO and apoE-KO × HL-KO mice, as well as LCAT-Tg and LCAT-Tg × HL-KO mice, chimeric for macrophage HL gene expression. Using in situ RNA hybridization, we demonstrated localized production of HL by donor macrophages in the artery wall. We found that expression of HL by macrophages enhances early aortic lesion formation in both apoE-KO and LCAT-Tg mice, without changing the plasma lipid profile, lipoprotein lipid composition, or HL and lipoprotein lipase activities. HL does, however, enhance oxidized LDL uptake by peritoneal macrophages. These combined data demonstrate that macrophage-derived HL significantly contributes to early aortic lesion formation in two independent mouse models and identify a novel mechanism, separable from the role of HL in plasma lipoprotein metabolism, by which HL modulates atherogenic risk in vivo.

I n fibrotic renal disease, elevated TGF-β and angiotensin II lead to increased plasminogen activator inhibitor type 1 (PAI-1). PAI-1 appears to reduce glomerular mesangial matrix turnover by inhibiting plasminogen activators, thereby decreasing plasmin generation and plasmin-mediated matrix degradation. We hypothesized that therapy with a mutant human PAI-1 (PAI-1R) that binds to matrix vitronectin but does not inhibit plasminogen activators, would enhance plasmin generation, increase matrix turnover, and decrease matrix accumulation in experimental glomerulonephritis. Three experimental groups included normal, untreated disease control, and PAI-1R–treated nephritic rats. Plasmin generation by isolated day 3 glomeruli was dramatically decreased by 69%, a decrease that was reversed 43% (P < 0.02) by in vivo PAI-1R treatment. At day 6, animals treated with PAI-1R showed significant reductions in proteinuria (48%, P < 0.02), glomerular staining for periodic acid–Schiff positive material (33%, P < 0.02), collagen I (28%, P < 0.01), collagen III (34%, P < 0.01), fibronectin (48%, P < 0.01), and laminin (41%, P < 0.01), and in collagen I (P < 0.01) and fibronectin mRNA levels (P < 0.02). Treatment did not alter overexpression of TGF-β1 and PAI-1 mRNAs, although TGF-β1 protein was significantly reduced. These observations strongly support our hypothesis that PAI-1R reduces glomerulosclerosis by competing with endogenous PAI-1, restoring plasmin generation, inhibiting inflammatory cell infiltration, decreasing local TGF-β1 concentration, and reducing matrix accumulation.

G alectin-3 is a member of a large family of animal lectins. This protein is expressed abundantly by macrophages, but its function in this cell type is not well understood. We have studied the effect of galectin-3 gene targeting on phagocytosis, a major function of macrophages. Compared with wild-type macrophages, galectin-3–deficient (gal3^–/–) cells exhibited reduced phagocytosis of IgG-opsonized erythrocytes and apoptotic thymocytes in vitro. In addition, gal3^–/– mice showed attenuated phagocytic clearance of apoptotic thymocytes by peritoneal macrophages in vivo. These mice also exhibited reduced IgG-mediated phagocytosis of erythrocytes by Kupffer cells in a murine model of autoimmune hemolytic anemia. Additional experiments indicate that extracellular galectin-3 does not contribute appreciably to the phagocytosis-promoting function of this protein. Confocal microscopic analysis of macrophages containing phagocytosed erythrocytes revealed localization of galectin-3 in phagocytic cups and phagosomes. Furthermore, gal3^–/– macrophages exhibited a lower degree of actin rearrangement upon Fcγ receptor crosslinkage. These results indicate that galectin-3 contributes to macrophage phagocytosis through an intracellular mechanism. Thus, galectin-3 may play an important role in both innate and adaptive immunity by contributing to phagocytic clearance of microorganisms and apoptotic cells.

T he critical role for ADP in arterial thrombogenesis was established by the clinical success of P2Y₁₂ antagonists, currently used at doses that block 40–50% of the P2Y₁₂ on platelets. This study was designed to determine the role of P2Y₁₂ in platelet thrombosis and how its complete absence affects the thrombotic process. P2Y₁₂-null mice were generated by a gene-targeting strategy. Using an in vivo mesenteric artery injury model and real-time continuous analysis of the thrombotic process, we observed that the time for appearance of first thrombus was delayed and that only small, unstable thrombi formed in P2Y₁₂^–/– mice without reaching occlusive size, in the absence of aspirin. Platelet adhesion to vWF was impaired in P2Y₁₂^–/– platelets. While adhesion to fibrinogen and collagen appeared normal, the platelets in thrombi from P2Y₁₂^–/– mice on collagen were less dense and less activated than their WT counterparts. P2Y₁₂^–/– platelet activation was also reduced in response to ADP or a PAR-4–activating peptide. Thus, P2Y₁₂ is involved in several key steps of thrombosis: platelet adhesion/activation, thrombus growth, and stability. The data suggest that more aggressive strategies of P2Y₁₂ antagonism will be antithrombotic without the requirement of aspirin cotherapy and may provide benefits even to the aspirin-nonresponder population.

H epatitis delta virus (HDV) can dramatically worsen liver disease in patients coinfected with hepatitis B virus (HBV). No effective medical therapy exists for HDV. The HDV envelope requires HBV surface antigen proteins provided by HBV. Once inside a cell, however, HDV can replicate its genome in the absence of any HBV gene products. In vitro, HDV virion assembly is critically dependent on prenyl lipid modification, or prenylation, of its nucleocapsid-like protein large delta antigen. To overcome limitations of current animal models and to test the hypothesis that pharmacologic prenylation inhibition can prevent the production of HDV virions in vivo, we established a convenient mouse-based model of HDV infection capable of yielding viremia. Such mice were then treated with the prenylation inhibitors FTI-277 and FTI-2153. Both agents were highly effective at clearing HDV viremia. As expected, HDV inhibition exhibited duration-of-treatment dependence. These results provide the first preclinical data supporting the in vivo efficacy of prenylation inhibition as a novel antiviral therapy with potential application to HDV and a wide variety of other viruses.

A lzheimer disease (AD) is characterized by the progressive deposition of the 42-residue amyloid β protein (Aβ) in brain regions serving memory and cognition. In animal models of AD, immunization with Aβ results in the clearance of Aβ deposits from the brain. However, a trial of vaccination with synthetic human Aβ1–42 in AD resulted in the development of meningoencephalitis in some patients. We measured cellular immune responses to Aβ in middle-aged and elderly healthy subjects and in patients with AD. A significantly higher proportion of healthy elderly subjects and patients with AD had strong Aβ-reactive T cell responses than occurred in middle-aged adults. The immunodominant Aβ epitopes in humans resided in amino acids 16–33. Epitope mapping enabled the identification of MHC/T cell receptor (TCR) contact residues. The occurrence of intrinsic T cell reactivity to the self-antigen Aβ in humans has implications for the design of Aβ vaccines, may itself be linked to AD susceptibility and course, and appears to be associated with the aging process.

T he cannabinoid receptor type 1 (CB1) and its endogenous ligands, the endocannabinoids, are involved in the regulation of food intake. Here we show that the lack of CB1 in mice with a disrupted CB1 gene causes hypophagia and leanness. As compared with WT (CB1^+/+) littermates, mice lacking CB1 (CB1^–/–) exhibited reduced spontaneous caloric intake and, as a consequence of reduced total fat mass, decreased body weight. In young CB1^–/– mice, the lean phenotype is predominantly caused by decreased caloric intake, whereas in adult CB1^–/– mice, metabolic factors appear to contribute to the lean phenotype. No significant differences between genotypes were detected regarding locomotor activity, body temperature, or energy expenditure. Hypothalamic CB1 mRNA was found to be coexpressed with neuropeptides known to modulate food intake, such as corticotropin-releasing hormone (CRH), cocaine-amphetamine–regulated transcript (CART), melanin-concentrating hormone (MCH), and prepro-orexin, indicating a possible role for endocannabinoid receptors within central networks governing appetite. CB1^–/– mice showed significantly increased CRH mRNA levels in the paraventricular nucleus and reduced CART mRNA levels in the dorsomedial and lateral hypothalamic areas. CB1 was also detected in epidydimal mouse adipocytes, and CB1-specific activation enhanced lipogenesis in primary adipocyte cultures. Our results indicate that the cannabinoid system is an essential endogenous regulator of energy homeostasis via central orexigenic as well as peripheral lipogenic mechanisms and might therefore represent a promising target to treat diseases characterized by impaired energy balance.

T he development and mechanisms of tolerance to allergens are poorly understood. Using the murine low zone tolerance (LZT) model, where contact hypersensitivity (CHS) is prevented by repeated topical low-dose applications of contact allergens, we show that LZT induction is IL-10 dependent. IL-10 is required for the generation of LZT effector cells, that is, CD8⁺ regulatory T cells. Only T cells from tolerized IL-10^+/+ mice or IL-10^–/– mice reconstituted with IL-10 during LZT induction adoptively transferred LZT to naive mice and prevented CHS, whereas T cells from IL-10^–/– mice failed to do so. The IL-10 required for normal LZT development is derived from lymph node CD4⁺ T cells, the only skin or lymph node cell population found to produce relevant amounts of IL-10 after tolerization. CD4⁺ T cells derived from IL-10^+/+ mice, but not from IL-10^–/– mice, allowed the induction of LZT in adoptively transferred T cell–deficient mice. Interestingly, IL-10 injections during tolerization greatly enhanced LZT responses in normal mice. Thus, the generation of CD8⁺ LZT effector T cells by CD4⁺ regulatory T cells via IL-10 may be a promising target of strategies aimed at preventing contact allergies and other harmful immune responses.

E pidemiologic studies demonstrate that long-term use of NSAIDs is associated with a reduced risk for the development of Alzheimer disease (AD). In this study, 20 commonly used NSAIDs, dapsone, and enantiomers of flurbiprofen were analyzed for their ability to lower the level of the 42-amino-acid form of amyloid β protein (Aβ42) in a human H4 cell line. Thirteen of the NSAIDs and the enantiomers of flurbiprofen were then tested in acute dosing studies in amyloid β protein precursor (APP) transgenic mice, and plasma and brain levels of Aβ and the drug were evaluated. These studies show that (a) eight FDA-approved NSAIDs lower Aβ42 in vivo, (b) the ability of an NSAID to lower Aβ42 levels in cell culture is highly predicative of its in vivo activity, (c) in vivo Aβ42 lowering in mice occurs at drug levels achievable in humans, and (d) there is a significant correlation between Aβ42 lowering and levels of ibuprofen. Importantly, flurbiprofen and its enantiomers selectively lower Aβ42 levels in broken cell γ-secretase assays, indicating that these compounds directly target the γ-secretase complex that generates Aβ from APP. Of the compounds tested, meclofenamic acid, racemic flurbiprofen, and the purified R and S enantiomers of flurbiprofen lowered Aβ42 levels to the greatest extent. Because R-flurbiprofen reduces Aβ42 levels by targeting γ-secretase and has reduced side effects related to inhibition of cyclooxygenase (COX), it is an excellent candidate for clinical testing as an Aβ42 lowering agent.

G riscelli syndrome (GS) is a rare autosomal recessive disorder that associates hypopigmentation, characterized by a silver-gray sheen of the hair and the presence of large clusters of pigment in the hair shaft, and the occurrence of either a primary neurological impairment or a severe immune disorder. Two different genetic forms, GS1 and GS2, respectively, account for the mutually exclusive neurological and immunological phenotypes. Mutations in the gene encoding the molecular motor protein Myosin Va (MyoVa) cause GS1 and the dilute mutant in mice, whereas mutations in the gene encoding the small GTPase Rab27a are responsible for GS2 and the ashen mouse model. We herein present genetic and functional evidence that a third form of GS (GS3), whose expression is restricted to the characteristic hypopigmentation of GS, results from mutation in the gene that encodes melanophilin (Mlph), the ortholog of the gene mutated in leaden mice. We also show that an identical phenotype can result from the deletion of the MYO5A F-exon, an exon with a tissue-restricted expression pattern. This spectrum of GS conditions pinpoints the distinct molecular pathways used by melanocytes, neurons, and immune cells in secretory granule exocytosis, which in part remain to be unraveled.