RanBP9 at the intersection between cofilin and Aß pathologies: rescue of neurodegenerative changes by RanBP9 reduction.

Molecular pathways underlying the neurotoxicity and production of amyloid ß protein (Aß) represent potentially promising therapeutic targets for Alzheimer's disease (AD). We recently found that overexpression of the scaffolding protein RanBP9 increases Aß production in cell lines and in transgenic mice while promoting cofilin activation and mitochondrial dysfunction. Translocation of cofilin to mitochondria and induction of cofilin-actin pathology require the activation/dephosphorylation of cofilin by Slingshot homolog 1 (SSH1) and cysteine oxidation of cofilin. In this study, we found that endogenous RanBP9 positively regulates SSH1 levels and mediates Aß-induced translocation of cofilin to mitochondria and induction of cofilin-actin pathology in cultured cells, primary neurons, and in vivo. Endogenous level of RanBP9 was also required for Aß-induced collapse of growth cones in immature neurons (days in vitro 9 (DIV9)) and depletion of synaptic proteins in mature neurons (DIV21). In vivo, amyloid precursor protein (APP)/presenilin-1 (PS1) mice exhibited 3.5-fold increased RanBP9 levels, and RanBP9 reduction protected against cofilin-actin pathology, synaptic damage, gliosis, and Aß accumulation associated with APP/PS1 mice. Brains slices derived from APP/PS1 mice showed significantly impaired long-term potentiation (LTP), and RanBP9 reduction significantly enhanced paired pulse facilitation and LTP, as well as partially rescued contextual memory deficits associated with APP/PS1 mice. Therefore, these results underscore the critical importance of endogenous RanBP9 not only in Aß accumulation but also in mediating the neurotoxic actions of Aß at the level of synaptic plasticity, mitochondria, and cofilin-actin pathology via control of the SSH1-cofilin pathway in vivo.

A randomised, phase II study of intetumumab, an anti-αv-integrin mAb, alone and with dacarbazine in stage IV melanoma.

BACKGROUND: α(v) integrins are involved in angiogenesis and melanoma tumourigenesis. Intetumumab (CNTO 95) is a fully human anti-α(v)-integrin monoclonal antibody. METHODS: In a multicentre, randomised, phase II study, stage IV melanoma patients were randomised 1:1:1:1 to 1000 mg m(-2) dacarbazine+placebo (n=32), 1000 mg m(-2) dacarbazine+10 mg kg(-1) intetumumab (n=32), 10 mg kg(-1) intetumumab (n=33), or 5 mg kg(-1) intetumumab (n=32) q3w. The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall survival (OS), objective response rate (ORR), adverse events, and pharmacokinetics. RESULTS: No statistically significant differences in efficacy were observed between groups. In the dacarbazine+placebo, dacarbazine+intetumumab, 10 mg kg(-1) intetumumab, and 5 mg kg(-1) intetumumab groups, median PFS was 1.8, 2.5, 1.4, and 1.4 months; median OS was 8, 11, 15, and 9.8 months; and ORR of complete+partial response was 10, 3, 6, and 0%. Nonlinear intetumumab pharmacokinetics and potential intetumumab-dacarbazine interactions were observed. Transient, asymptomatic, nonrecurring, grade 1-2, uveitic reactions that resolved spontaneously or with topical steroids were seen in 22-30% of intetumumab-treated patients. Low-grade infusion-reaction symptoms (headache, fatigue, nausea, vomiting, fever, chills) were observed, as expected, in 16-73% of dacarbazine-treated patients. No intetumumab-related myelosuppression, laboratory/electrocardiogram abnormalities, or deaths occurred. CONCLUSION: With its favourable safety profile and a nonsignificant trend towards improved OS, intetumumab merits further investigation in advanced melanoma.

In vitro evaluation of an enhanced human serum amyloid A (SAA2) promoter-regulated soluble TNF receptor fusion protein for anti-inflammatory gene therapy.

Tumour necrosis factor (TNF)-alpha contributes to the pathogenesis of many inflammatory diseases. Recombinant soluble TNF receptor fusion proteins (sTNFR:Ig) are potent TNF antagonists, both in vitro and in vivo. The concentration of serum amyloid A (SAA) increases by up to 1000-fold during inflammation, largely owing to cytokine-driven transcriptional upregulation. A reporter plasmid, comprising the proximal 0.7 kb of the human SAA2 promoter fused to a luciferase gene, was used in transient transfection experiments in human HepG2 hepatoma cells to assess the quantitative and qualitative TNF antagonist properties of a construct in which sTNFR:Ig synthesis is under the control of a chimera of the SAA2 promoter and a tat/HIV element. The SAA2-tat/HIV-sTNFR:Ig construct retained the fine-tuned cytokine responsiveness of the SAA2 promoter, while exhibiting the quantitatively enhanced level of protein expression conferred by the tat/HIV element. It produced a biologically significant TNF inhibition that was at least as strong as that achieved using a CMV promoter-driven sTNFR:Ig construct. There was a dose- and time-dependent relationship between the pro-inflammatory cytokine used, and the generation of TNF antagonist activity by SAA2-tat/HIV-sTNFR:Ig. Although sTNFR:Ig protein can be induced by either TNF-alpha or interleukin (IL)-1beta, its antagonist activity is limited to the former cytokine. The SAA2-tat/HIV-sTNFR:Ig construct, and derivatives thereof, may therefore be ideally suited to gene therapy applications that require the local production of potent and specific immune modifiers only when there is active pathology. It may consequently be of particular use in the future treatment of diseases such as rheumatoid arthritis.

Serum amyloid A, the major vertebrate acute-phase reactant.

The serum amyloid A (SAA) family comprises a number of differentially expressed apolipoproteins, acute-phase SAAs (A-SAAs) and constitutive SAAs (C-SAAs). A-SAAs are major acute-phase reactants, the in vivo concentrations of which increase by as much as 1000-fold during inflammation. A-SAA mRNAs or proteins have been identified in all vertebrates investigated to date and are highly conserved. In contrast, C-SAAs are induced minimally, if at all, during the acute-phase response and have only been found in human and mouse. Although the liver is the primary site of synthesis of both A-SAA and C-SAA, extrahepatic production has been reported for most family members in most of the mammalian species studied. In vitro, the dramatic induction of A-SAA mRNA in response to pro-inflammatory stimuli is due largely to the synergistic effects of cytokine signaling pathways, principally those of the interleukin-1 and interleukin-6 type cytokines. This induction can be enhanced by glucocorticoids. Studies of the A-SAA promoters in several mammalian species have identified a range of transcription factors that are variously involved in defining both cytokine responsiveness and cell specificity. These include NF-kappaB, C/EBP, YY1, AP-2, SAF and Sp1. A-SAA is also post-transcriptionally regulated. Although the precise role of A-SAA in host defense during inflammation has not been defined, many potential clinically important functions have been proposed for individual SAA family members. These include involvement in lipid metabolism/transport, induction of extracellular-matrix-degrading enzymes, and chemotactic recruitment of inflammatory cells to sites of inflammation. A-SAA is potentially involved in the pathogenesis of several chronic inflammatory diseases: it is the precursor of the amyloid A protein deposited in amyloid A amyloidosis, and it has also been implicated in the pathogenesis of atheroscelerosis and rheumatoid arthritis.

The kinetics and magnitude of the synergistic activation of the serum amyloid A promoter by IL-1 beta and IL-6 is determined by the order of cytokine addition.

Human serum amyloid A protein (A-SAA) is a major hepatic acute-phase protein, the concentration of which increases by up to 1000-fold during inflammation. This induction is primarily due to synergistic transcriptional up-regulation by pro-inflammatory cytokines, principally interleukin (IL)-1 and IL-6. Using HepG2 hepatoma cells transfected with pGL2-SAA2pt, a cytokine-responsive human SAA2 promoter/luciferase reporter gene construct, we show that stimulation with IL-1 beta prior to IL-6 is essential for maximal synergistic transcriptional induction of the SAA2 gene. The reciprocal treatment, i.e. stimulation of the promoter with IL-6 before IL-1 beta results in significantly less synergistic activation of the SAA2 promoter. These findings strongly suggest that in vitro studies of acute-phase-protein induction using combinations of cytokines should be designed to reflect the chronology of their participation in the cytokine cascade.

Expression of a biologically active recombinant mouse IL-1 receptor antagonist and its use in vivo to modulate aspects of the acute phase response.

Recombinant mouse IL-1receptor antagonist protein (rmIL-1ra) was expressed in Escherichia coli. In vivo administration of rmIL-1ra, in a casein-induced murine model of acute inflammation, completely abolished the hepatic induction of the mRNAs specifying serum amyloid A1 (A-SAA1) and A-SAA2 for up to 12 h, indicating that hepatic A-SAA mRNA synthesis is totally IL-1 driven. A-SAA protein, however, was present in the serum of rmIL-1ra-treated casein-stimulated mice (although at lower levels than in untreated casein-stimulated mice) at 12 h indicating that extrahepatic A-SAA synthesis is driven in part by factors acting independently of IL-1. Hepatic mRNA levels of the other mouse acute phase reactants (APRs), serum amyloid P component, C-reactive protein, alpha1-acid glycoprotein, and C3 were also induced with casein after 12 h, as were serum protein levels of SAP and C3. These inductions were only partially inhibited by rmIL-1ra, indicating that hepatic expression of the latter APRs (unlike that of A-SAA) is driven partly by IL-1 and partly by factors acting independently of IL-1. Hepatic mRNA levels of the negative APRs apolipoprotein A-I and serum albumin were down-regulated 12 h after casein stimulation. rmIL-1ra partially restored serum albumin mRNA levels but not apo A-I mRNA levels, indicating differential regulation of these negative APRs. The rmIL-1ra will be useful in studies of IL-1-mediated gene regulation in murine models of inflammation.

Use of the acute phase serum amyloid A2 (SAA2) gene promoter in the analysis of pro- and anti-inflammatory mediators: differential kinetics of SAA2 promoter induction by IL-1 beta and TNF-alpha compared to IL-6.

A cytokine responsive construct, pGL2-SAA2pt, was generated by cloning the acute phase promoter of human serum amyloid A2 (SAA2) upstream of a luciferase reporter gene. The construct responds to the inflammatory mediators MoCM, IL-1 beta, TNF-alpha, and IL-6 in a manner that closely mimics the response of the endogenous SAA2 gene to such stimuli: i.e. single treatments induce transcriptional activation by IL-1 beta and TNF-alpha to a greater extent than by IL-6 at 12-24 h. However, timecourse experiments show that the kinetics of induction generated by IL-1 beta and TNF-alpha are quite distinct from IL-6, IL-6 having a much greater effect at 3-6 h. IL-1 beta and TNF-alpha synergize with IL-6 to give a 10-fold increase in transcriptional readout over single cytokine treatments. The kinetics of this synergistic response resembles that generated by IL-6 alone. The IL-1 receptor antagonist, hIL-1ra, can specifically block the IL-1 beta driven transcriptional activation of pGL2-SAA2pt, but not that driven by TNF-alpha or IL-6. Furthermore, in synergistic cytokine combinations, it blocks only the IL-1 beta driven component indicating that the effect is biological and not attributable to toxicity. Consequently assays utilizing pGL2-SAA2pt will be useful both for the investigation of the kinetics of inflammatory signalling in a cytokine specific manner, and for the evaluation of the pro- and anti-inflammatory properties of novel natural and synthetic molecules.

Acute phase proteins in salmonids: evolutionary analyses and acute phase response.

Inflammation induces dramatic changes in the biosynthetic profile of the liver, leading to increased serum concentrations of positive acute phase (AP) proteins and decreased concentrations of negative AP proteins. Serum amyloid A (SAA) and the pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP) are major AP proteins: their serum levels can rise by 1000-fold, indicating that they play a critical role in defense and/or the restoration of homeostasis. We have cloned SAA and a SAP-like pentraxin from salmonid fish species. The salmonid SAA shares approximately 70% amino acid identity with mammalian AP SAA. When salmonids are challenged with an AP stimulus, i.e., Aeromonas salmonicida, SAA responds dramatically as a major AP reactant. The salmonid pentraxin shows approximately 40% amino acid identity to both mammalian SAP and CRP. Evolutionary analysis suggests the presence of only a single such protein in teleosts and lower animal species. Surprisingly, the salmonid pentraxin behaves as a negative AP reactant, reminiscent of the SAP-like Syrian hamster female protein, in that hepatic mRNA concentrations decline to 50% of prestimulus levels. This study reinforces the hypothesis that SAA induction is an essential and universal feature of the vertebrate AP response and that it represents part of an ancient host defense system. Conversely, the species-dependent heterogeneity of pentraxin expression during the vertebrate AP response supports the possibility that its most important ancestral (and perhaps present) function is not related to its AP behavior.

Expression and regulation of constitutive and acute phase serum amyloid A mRNAs in hepatic and non-hepatic cell lines.

'Acute phase' and 'constitutive' SAA (A-SAA and C-SAA, respectively) mRNA levels were measured in hepatic and non-hepatic cell lines after treatment with monocyte conditioned medium (MoCM), with or without dexamethasone (Dex). A-SAA mRNAs were detected in MoCM-treated hepatoma cell lines (PLC/PRF/5, HuH7, HepG2, and Hep3B), a fibroblast cell line (MRC5), six epithelial cell lines (RT4/ 31, SW13, Hela Ohio, HCT-8, CaCo2, and KB), and an endothelial cell line ECV304. In KB cells, Dex alone caused a dramatic increase in A-SAA mRNA levels. C-SAA was detected in all hepatic and non-hepatic cell lines. Two differentially regulated size classes of C-SAA mRNA were detected in the hepatoma cell lines. A-SAA mRNA levels were measured in ECV304 cells treated with IL-1 beta, IL-6, TNF alpha and Dex, in various combinations, and revealed different profiles to those seen for hepatic cells. The extent of polyadenylation of A-SAA mRNA in ECV304 and KB cells differed whereas the polyadenylation of C-SAA mRNA remained constant. These data suggest that the parameters that determine the steady state mRNA levels and post-transcriptional regulation of A-SAA and C-SAA mRNAs are different and are cell type specific.

Wallaby serum amyloid A protein: cDNA cloning, sequence and evolutionary analysis.

A serum amyloid A (SAA) clone was isolated from a Tammar wallaby cDNA library, the most distantly related mammalian species for which an SAA has been described to date. The clone predicts a premolecule of 127 amino acids with good homology to other mammalian SAAs, and consists of an 18 residue leader peptide and a mature protein of 109 amino acids. Evolutionary analysis at both the protein and nucleotide level indicate that the wallaby SAA clone clusters with the acute phase SAAs. However, as the SAA superfamily has undergone concerted evolution it is not possible to determine at this point which acute phase SAA it is most like. The grouping of wallaby SAA inside the acute phase SAA cluster demonstrates that at least some of the duplication events giving rise to multiple acute phase genes occurred prior to the divergence of the eutherian and metatherian mammals.

Evolution of the serum amyloid A (SAA) protein superfamily.

The serum amyloid A (SAA) superfamily comprises a number of genes and proteins characterized from a range of mammalian species. The majority of members described to date are dramatically induced during the acute-phase response, suggesting an important short-term beneficial role in the response to tissue injury and inflammation. However, important disease associations have also been proposed for certain SAAs during chronic inflammation. The nomenclature of many of the superfamily members has been the result of comparisons with previously reported sequences implying disease association and/or functional relatedness between such members. The evolutionary relationships of the SAA superfamily members have been investigated by comparisons at both the amino acid and the nucleotide level. The results indicate that all members of the superfamily within a species have been undergoing concerted evolution. This has important implications in ascribing functions and disease associations to individual SAA superfamily members and indicates that designations should not be based on the extent of amino acid identity alone but should be made only following direct experimental observation of the proteins themselves.

The mouse interleukin 1 receptor antagonist protein: gene structure and regulation in vitro.

The interleukin 1 receptor antagonist (IL-1ra) protein is an inhibitor of the pro-inflammatory cytokine interleukin 1. We have sequenced the mouse gene encoding the monocyte form of IL-1ra (IL-1rn) and compared it with the sequence of the human homologue. In addition to high levels of similarity between the coding regions of the two genes, portions of the introns show surprisingly high levels of identity. In order to develop an in vitro model system to investigate the regulation of IL-1ra induction, three differently responding mouse macrophage cell lines were stimulated with lipopolysaccharide. The kinetics and magnitude of IL-1ra mRNA accumulation was cell-line specific indicating that IL-1ra synthesis in response to inducing agents varies according to the phenotype of the cell. Analysis of the relative transcription rate and the half life of the mouse IL-1ra mRNA indicate that IL-1ra mRNA accumulation in macrophages following LPS treatment is due primarily to an increase in transcription rate rather than to increased stability.

A constitutively expressed serum amyloid A protein gene (SAA4) is closely linked to, and shares structural similarities with, an acute-phase serum amyloid A protein gene (SAA2).

The acute-phase reactant serum amyloid A (SAA) is a polymorphic apolipoprotein encoded by a family of highly homologous and closely linked genes: SAA1, SAA2, and SAA3. We have isolated a human genomic cosmid clone containing the gene encoding a fourth, constitutively expressed member of the human SAA superfamily, C-SAA, together with an SAA2*2 (SAA2 beta) gene. The gene encoding C-SAA shares the same 5' to 3' orientation as SAA2*2 and has the characteristic four-exon structure of the other members of the SAA superfamily. The exons of the gene encoding C-SAA share only limited sequence identity with those of SAA1, SAA2, and SAA3; they specify an mRNA, represented by the CS-1 cDNA reported previously by us, which is expressed at low levels (relative to the acute-phase SAAs) in normal and acute-phase liver. The gene encoding C-SAA is located 9 kb downstream of SAA2*2 and therefore occupies the locus that has been identified as containing the SAA4 gene.