CD28 homodimer interface mimetic peptide acts as a preventive and therapeutic agent in models of severe bacterial sepsis and gram-negative bacterial peritonitis.

BACKGROUND: Severe gram-negative bacterial infections and sepsis are major causes of morbidity and mortality. Dysregulated, excessive proinflammatory cytokine expression contributes to the pathogenesis of sepsis. A CD28 mimetic peptide (AB103; previously known as p2TA) that attenuates CD28 signaling and T-helper type 1 cytokine responses was tested for its ability to increase survival in models of polymicrobial infection and gram-negative sepsis. METHODS: Mice received AB103, followed by an injection of Escherichia coli 0111:B4 lipopolysaccharide (LPS); underwent induction E. coli 018:K1 peritonitis induction, followed by treatment with AB103; or underwent cecal ligation and puncture (CLP), followed by treatment with AB103. The effects of AB103 on factors associated with and the lethality of challenge infections were analyzed. RESULTS: AB103 strongly attenuated induction of tumor necrosis factor α and interleukin 6 (IL-6) by LPS in human peripheral blood mononuclear cells. Receipt of AB103 following intraperitoneal injection of LPS resulted in survival among 73% of CD1 mice (11 of 15), compared with 20% of controls (3 of 15). Suboptimal doses of antibiotic alone protected 20% of mice (1 of 5) from E. coli peritonitis, whereas 100% (15 of 15) survived when AB103 was added 4 hours following infection. Survival among mice treated with AB103 12 hours after CLP was 100% (8 of 8), compared with 17% among untreated mice (1 of 6). In addition, receipt of AB103 12 hours after CLP attenuated inflammatory cytokine responses and neutrophil influx into tissues and promoted bacterial clearance. Receipt of AB103 24 hours after CLP still protected 63% of mice (5 of 8). CONCLUSIONS: Single-dose AB103 reduces mortality in experimental models of polymicrobial and gram-negative bacterial infection and sepsis, warranting further studies of this agent in clinical trials.

A peptide antagonist of CD28 signaling attenuates toxic shock and necrotizing soft-tissue infection induced by Streptococcus pyogenes.

Staphylococcus aureus and group A Streptococcus pyogenes (GAS) express superantigen (SAg) exotoxin proteins capable of inducing lethal shock. To induce toxicity, SAgs must bind not only to the major histocompatibility complex II molecule of antigen-presenting cells and the variable ß chain of the T-cell receptor but also to the dimer interface of the T-cell costimulatory receptor CD28. Here, we show that the CD28-mimetic peptide AB103 (originally designated "p2TA") protects mice from lethal challenge with streptococcal exotoxin A, as well as from lethal GAS bacterial infection in a murine model of necrotizing soft-tissue infection. Administration of a single dose of AB103 increased survival when given up to 5 hours after infection, reduced inflammatory cytokine expression and bacterial burden at the site of infection, and improved muscle inflammation in a dose-dependent manner, without compromising cellular and humoral immunity. Thus, AB103 merits further investigation as a potential therapeutic in SAg-mediated necrotizing soft-tissue infection.

A novel human heparanase splice variant, T5, endowed with protumorigenic characteristics.

Heparanase is a mammalian endo-beta-d-glucuronidase that can cleave heparan sulfate side chains, an activity strongly implicated in tumor cell dissemination. The current study aimed to identify and characterize heparanase splice variants. LEADS, Compugen's alternative splicing modeling platform (Compugen, Tel Aviv, Israel), was used to search for splice variants in silico; tumor-derived cell lines (i.e., CAG myeloma) and tumor biopsies were utilized to validate T5 expression in vivo; signaling (i.e., Src phosphorylation) was evaluated following T5 gene silencing or overexpression and correlated with cell proliferation, colony formation, and tumor xenograft development. A novel spliced form of human heparanase, termed T5, was identified. In this splice variant, 144 bp of intron 5 are joined with exon 4, which results in a truncated, enzymatically inactive protein. T5 overexpression resulted in increased cell proliferation and larger colonies in soft agar, mediated by Src activation. Furthermore, T5 overexpression markedly enhanced tumor xenograft development. T5 expression is up-regulated in 75% of human renal cell carcinoma biopsies examined, which suggests that this splice variant is clinically relevant. Controls included cells overexpressing wild-type heparanase or an empty plasmid and normal-looking tissue adjacent the carcinoma lesion. T5 is a novel functional splice variant of human heparanase endowed with protumorigenic characteristics.-Barash, U., Cohen-Kaplan, V., Arvatz, G., Gingis-Velitski, S., Levy-Adam, F., Nativ, O., Shemesh, R., Ayalon-Sofer, M., Ilan, N., Vlodavsky, I. A novel human heparanase splice variant, T5, endowed with protumorigenic characteristics.

Prevention of bleomycin-induced pulmonary fibrosis by a novel antifibrotic peptide with relaxin-like activity.

Pulmonary fibrosis is a progressive and lethal lung disease characterized by accumulation of extracellular matrix and loss of pulmonary function. No cure exists for this pathologic condition, and current treatments often fail to slow its progression or relieve its symptoms. Relaxin was previously shown to induce a matrix-degrading phenotype in human lung fibroblasts in vitro and to inhibit pulmonary fibrosis in vivo. A novel peptide that targets the relaxin RXFP1/LGR7 receptor was recently identified using our computational platform designed to predict novel G protein-coupled receptor peptide agonists. In this study, we examined the antifibrotic properties of this novel peptide, designated CGEN25009, in human cell-based assays and in a murine model of bleomycin-induced pulmonary fibrosis. Similar to relaxin, CGEN25009 was found to have an inhibitory effect on transforming growth factor-ß1-induced collagen deposition in human dermal fibroblasts and to enhance MMP-2 expression. The peptide's biological activity was also similar to relaxin in generating cellular stimulation of cAMP, cGMP, and NO in the THP-1 human cell line. In vivo, 2-week administration of CGEN25009 in a preventive or therapeutic mode (i.e., concurrently with or 7 days after bleomycin treatment, respectively) caused a significant reduction in lung inflammation and injury and ameliorated adverse airway remodeling and peribronchial fibrosis. The results of this study indicate that CGEN25009 displays antifibrotic and anti-inflammatory properties and may offer a new therapeutic option for the treatment of pulmonary fibrosis.

Separation of roles of Zip1 in meiosis revealed in heterozygous mutants of Saccharomyces cerevisiae.

Synapsis of homologs during meiotic prophase I is associated with a protein complex built along the bivalents--the synaptonemal complex (SC). Mutations in the SC-component gene ZIP1 diminish SC formation, leading to reduced recombination levels and low spore viability. Here we show that in SK1 strains heterozygous for a deletion of ZIP1 in certain regions meiotic interference are impaired with no decrease in recombination levels. The extent of synapsis is over all reduced and NDJ levels of a large endogenous chromosome and of artificial chromosomes (YACs) rise to twice the level of wild type strains. A substantial proportion of mis-segregating YACs had undergone crossing over. This demonstrates that different functions of Zip1 display differential sensitivities to changes in expression levels.

A novel peptide agonist of formyl-peptide receptor-like 1 (ALX) displays anti-inflammatory and cardioprotective effects.

Activation of the formyl-peptide receptor-like (FPRL) 1 pathway has recently gained high recognition for its significance in therapy of inflammatory diseases. Agonism at FPRL1 affords a beneficial effect in animal models of acute inflammatory conditions, as well as in chronic inflammatory diseases. TIPMFVPESTSKLQKFTSWFM-amide (CGEN-855A) is a novel 21-amino acid peptide agonist for FPRL1 and also activates FPRL2. CGEN-855A was discovered using a computational platform designed to predict novel G protein-coupled receptor peptide agonists cleaved from secreted proteins by convertase proteolysis. In vivo, CGEN-855A displays anti-inflammatory activity manifested as 50% inhibition of polymorphonuclear neutrophil (PMN) recruitment to inflamed air pouch and provides protection against ischemia-reperfusion-mediated injury to the myocardium in both murine and rat models (36 and 25% reduction in infarct size, respectively). Both these activities are accompanied by inhibition of PMN recruitment to the injured organ. The secretion of inflammatory cytokines, including interleukin (IL)-6, IL-1beta, and tumor necrosis factor-alpha, was not affected upon incubation of human peripheral blood mononuclear cells with CGEN-855A, whereas IL-8 secretion was elevated up to 2-fold upon treatment with the highest CGEN-855A dose only. Collectively, these new data support a potential role for CGEN-855A in the treatment of reperfusion-mediated injury and in other acute and chronic inflammatory conditions.

Systematic identification of abundant A-to-I editing sites in the human transcriptome.

RNA editing by members of the ADAR (adenosine deaminases acting on RNA) family leads to site-specific conversion of adenosine to inosine (A-to-I) in precursor messenger RNAs. Editing by ADARs is believed to occur in all metazoa, and is essential for mammalian development. Currently, only a limited number of human ADAR substrates are known, whereas indirect evidence suggests a substantial fraction of all pre-mRNAs being affected. Here we describe a computational search for ADAR editing sites in the human transcriptome, using millions of available expressed sequences. We mapped 12,723 A-to-I editing sites in 1,637 different genes, with an estimated accuracy of 95%, raising the number of known editing sites by two orders of magnitude. We experimentally validated our method by verifying the occurrence of editing in 26 novel substrates. A-to-I editing in humans primarily occurs in noncoding regions of the RNA, typically in Alu repeats. Analysis of the large set of editing sites indicates the role of editing in controlling dsRNA stability.

Evolutionarily conserved human targets of adenosine to inosine RNA editing.

A-to-I RNA editing by ADARs is a post-transcriptional mechanism for expanding the proteomic repertoire. Genetic recoding by editing was so far observed for only a few mammalian RNAs that are predominantly expressed in nervous tissues. However, as these editing targets fail to explain the broad and severe phenotypes of ADAR1 knockout mice, additional targets for editing by ADARs were always expected. Using comparative genomics and expressed sequence analysis, we identified and experimentally verified four additional candidate human substrates for ADAR-mediated editing: FLNA, BLCAP, CYFIP2 and IGFBP7. Additionally, editing of three of these substrates was verified in the mouse while two of them were validated in chicken. Interestingly, none of these substrates encodes a receptor protein but two of them are strongly expressed in the CNS and seem important for proper nervous system function. The editing pattern observed suggests that some of the affected proteins might have altered physiological properties leaving the possibility that they can be related to the phenotypes of ADAR1 knockout mice.

Follow the leader: preference for specific amino acids directly following the initial methionine in proteins of different organisms.

It is well established that the vast majority of proteins of all taxonomical groups and species are initiated by an AUG codon, translated into the amino acid methionine (Met). Many attempts were made to evaluate the importance of the sequences surrounding the initiation codon, mostly focusing on the RNA sequence. However, the role and importance of the amino acids following the initiating Met residue were rarely investigated, mostly in bacteria and fungi. Herein, we computationally examined the protein sequences of all major taxonomical groups represented in the Swiss-Prot database, and evaluated the preference of each group to specific amino acids at the positions directly following the initial Met. The results indicate that there is a species-specific preference for the second amino acid of the majority of protein sequences. Interestingly, the preference for a certain amino acid at the second position changes throughout evolution from lysine in prokaryotes, through serine in lower eukaryotes, to alanine in higher plants and animals.

Activation of relaxin-related receptors by short, linear peptides derived from a collagen-containing precursor.

In a screening effort based on algorithmic predictions for novel G-protein-coupled receptor (GPCR) peptide activators, we were able to identify and examine two novel peptides (P59 and P74) which are short, linear, and derived from a natural, previously unidentified precursor protein containing a collagen-like repeat. Both peptides seemed to show an apparent cAMP-related effect on CHO-K1 cells transiently transfected with either LGR7 or LGR8, usually after treatment with cAMP-generating forskolin, compared to the same cells treated with forskolin plus relaxin. This activation was not found for the relaxin-3 receptor (GPR135). In a set of follow-up experiments, both peptides were found to stimulate cAMP production, mostly upon initial stimulation of cAMP production by 5 micro M forskolin in cells transfected with either LGR7 or LGR8. In a dye-free cell impedance GPCR activation assay, we were able to show that these peptides were also able to activate a cellular response mediated by these receptors. Although untransfected CHO-K1 cells showed some cellular activation by both relaxin and at least one of our newly discovered peptides, both LGR7- and LGR8-transfected cells showed a stronger response, indicating stimulation of a cellular pathway through activation of these receptors. In conclusion, we were able to show that these newly discovered peptides, which have no similarity to any member of the relaxin-insulin-like peptide family, are potential ligands for the relaxin-related family of receptors and as such might serve as novel candidates for relaxin-related therapeutic indications. Both peptides are linear and were found to be active after being chemically synthesized.

Discovery and validation of novel peptide agonists for G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) represent an important group of targets for pharmaceutical therapeutics. The completion of the human genome revealed a large number of putative GPCRs. However, the identification of their natural ligands, and especially peptides, suffers from low discovery rates, thus impeding development of therapeutics based on these potential drug targets. We describe the discovery of novel GPCR ligands encrypted in the human proteome. Hundreds of potential peptide ligands were predicted by machine learning algorithms. In vitro screening of selected 33 peptides on a set of 152 GPCRs, including a group of designated orphan receptors, was conducted by intracellular calcium measurements and cAMP assays. The screening revealed eight novel peptides as potential agonists that specifically activated six different receptors in a dose-dependent manner. Most of the peptides showed distinct stimulatory patterns targeted at designated and orphan GPCRs. Further analysis demonstrated a significant in vivo effect for one of the peptides in a mouse inflammation model.

Genomic fossils as a snapshot of the human transcriptome.

Processed pseudogenes (PPGs) are cDNA sequences that were generated through reverse transcription of mature, spliced mRNAs and have subsequently been reinserted at a new genomic location. These cDNA sequences are usually no longer transcribed and are considered "dead on arrival." Here we show that PPGs can be used to generate a map of the transcriptome. By analyzing thousands of human PPGs, we were able to discover hundreds of transcript variants so far unidentified. An experimental verification of a subset of these variants by RT-PCR indicates that most of them are still active in the human transcriptome. Furthermore, we demonstrate that PPGs can enable the identification of ancient splice variants that were expressed ancestrally but are now extinct. Our results show that the genome itself carries a "virtual cDNA library" that can readily be used to analyze both present and ancestral transcripts. Our approach can be applied to sequenced metazoan genomes to computationally annotate splicing variation even when expressed sequences are unavailable.

Transcription-mediated gene fusion in the human genome.

Transcription of a gene usually ends at a regulated termination point, preventing the RNA-polymerase from reading through the next gene. However, sporadic reports suggest that chimeric transcripts, formed by transcription of two consecutive genes into one RNA, can occur in human. The splicing and translation of such RNAs can lead to a new, fused protein, having domains from both original proteins. Here, we systematically identified over 200 cases of intergenic splicing in the human genome (involving 421 genes), and experimentally demonstrated that at least half of these fusions exist in human tissues. We showed that unique splicing patterns dominate the functional and regulatory nature of the resulting transcripts, and found intergenic distance bias in fused compared with nonfused genes. We demonstrate that the hundreds of fused genes we identified are only a subset of the actual number of fused genes in human. We describe a novel evolutionary mechanism where transcription-induced chimerism followed by retroposition results in a new, active fused gene. Finally, we provide evidence that transcription-induced chimerism can be a mechanism contributing to the evolution of protein complexes.

A non-EST-based method for exon-skipping prediction.

It is estimated that between 35% and 74% of all human genes can undergo alternative splicing. Currently, the most efficient methods for large-scale detection of alternative splicing use expressed sequence tags (ESTs) or microarray analysis. As these methods merely sample the transcriptome, splice variants that do not appear in deeply sampled tissues have a low probability of being detected. We present a new method by which we can predict that an internal exon is skipped (namely whether it is a cassette-exon) merely based on its naked genomic sequence and on the sequence of its mouse ortholog. No other data, such as ESTs, are required for the prediction. Using our method, which was experimentally validated, we detected hundreds of novel splice variants that were not detectable using ESTs. We show that a substantial fraction of the splice variants in the human genome could not be identified through current human EST or cDNA data.

Liver micro-organs transcribe albumin and clotting factors and increase survival of 92% hepatectomized rats.

BACKGROUND/AIMS: Currently there is no effective non-surgical therapy for most patients with fulminant or end stage chronic liver disease. METHODS: We have prepared rat liver micro-organs (LMOs), which preserve the liver micro-architecture and ensure that no cell is more than 150 microm away from a source of nutrients and gases. The function of LMOs has been evaluated in vitro and in a new extra-corporeal liver device termed aLIVE in which LMOs are exposed to liver-like hemodynamic conditions. RESULTS: In vitro LMOs maintain normal physiological and biochemical functions including oxygen consumption, glucose metabolism, conversion of ammonia to urea, secretion of albumin and de novo transcription of genes coding for albumin and clotting factors. Inside the aLIVE bioreactor, LMOs also display sustained oxygen consumption, glucose metabolism and transcription of albumin and clotting factors IX and X, when connected both to normal and to 92% hepatectomized rats. Survival of 92% hepatectomized rats was 40% longer following a single 4-h treatment with aLIVE, compared to untreated animals. CONCLUSIONS: An extra-corporeal liver device, aLIVE, which provides key liver functions, has been developed. When tested in 92% hepatectomized rats, aLIVE improved the clinical condition and significantly increased survival time of the treated rats.