CBLB613: a TLR 2/6 agonist, natural lipopeptide of Mycoplasma arginini , as a novel radiation countermeasure.

To date, there are no safe and effective drugs available for protection against ionizing radiation damage. Therefore, a great need exists to identify and develop non-toxic agents that will be useful as radioprotectors or postirradiation therapies under a variety of operational scenarios. We have developed a new pharmacological agent, CBLB613 (a naturally occurring Mycoplasma-derived lipopeptide ligand for Toll-like receptor 2/6), as a novel radiation countermeasure. Using CD2F1 mice, we investigated CBLB613 for toxicity, immunogenicity, radioprotection, radiomitigation and pharmacokinetics. We also evaluated CBLB613 for its effects on cytokine induction and radiation-induced cytopenia in unirradiated and irradiated mice. The no-observable-adverse-effect level of CBLB613 was 1.79 mg/kg and 1 mg/kg for single and repeated doses, respectively. CBLB613 significantly protected mice against a lethal dose of (60)Co γ radiation. The dose reduction factor of CBLB613 as a radioprotector was 1.25. CBLB613 also mitigated the effects of (60)Co γ radiation on survival in mice. In both irradiated and unirradiated mice, the drug stimulated induction of interleukin-1ß (IL-1ß), IL-6, IL-10, IL-12, keratinocyte-derived chemokine, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-1α. CBLB613 also reduced radiation-induced cytopenia and increased bone marrow cellularity in irradiated mice. Our immunogenicity study demonstrated that CBLB613 is not immunogenic in mice, indicating that it could be developed as a radioprotector and radiomitigator for humans against the potentially lethal effects of radiation exposure.

Prevention and mitigation of acute radiation syndrome in mice by synthetic lipopeptide agonists of Toll-like receptor 2 (TLR2).

Bacterial lipoproteins (BLP) induce innate immune responses in mammals by activating heterodimeric receptor complexes containing Toll-like receptor 2 (TLR2). TLR2 signaling results in nuclear factor-kappaB (NF-κB)-dependent upregulation of anti-apoptotic factors, anti-oxidants and cytokines, all of which have been implicated in radiation protection. Here we demonstrate that synthetic lipopeptides (sLP) that mimic the structure of naturally occurring mycoplasmal BLP significantly increase mouse survival following lethal total body irradiation (TBI) when administered between 48 hours before and 24 hours after irradiation. The TBI dose ranges against which sLP are effective indicate that sLP primarily impact the hematopoietic (HP) component of acute radiation syndrome. Indeed, sLP treatment accelerated recovery of bone marrow (BM) and spleen cellularity and ameliorated thrombocytopenia of irradiated mice. sLP did not improve survival of irradiated TLR2-knockout mice, confirming that sLP-mediated radioprotection requires TLR2. However, sLP was radioprotective in chimeric mice containing TLR2-null BM on a wild type background, indicating that radioprotection of the HP system by sLP is, at least in part, indirect and initiated in non-BM cells. sLP injection resulted in strong transient induction of multiple cytokines with known roles in hematopoiesis, including granulocyte colony-stimulating factor (G-CSF), keratinocyte chemoattractant (KC) and interleukin-6 (IL-6). sLP-induced cytokines, particularly G-CSF, are likely mediators of the radioprotective/mitigative activity of sLP. This study illustrates the strong potential of LP-based TLR2 agonists for anti-radiation prophylaxis and therapy in defense and medical scenarios.

The complete genome of hyperthermophile Methanopyrus kandleri AV19 and monophyly of archaeal methanogens.

We have determined the complete 1,694,969-nt sequence of the GC-rich genome of Methanopyrus kandleri by using a whole direct genome sequencing approach. This approach is based on unlinking of genomic DNA with the ThermoFidelase version of M. kandleri topoisomerase V and cycle sequencing directed by 2'-modified oligonucleotides (Fimers). Sequencing redundancy (3.3x) was sufficient to assemble the genome with less than one error per 40 kb. Using a combination of sequence database searches and coding potential prediction, 1,692 protein-coding genes and 39 genes for structural RNAs were identified. M. kandleri proteins show an unusually high content of negatively charged amino acids, which might be an adaptation to the high intracellular salinity. Previous phylogenetic analysis of 16S RNA suggested that M. kandleri belonged to a very deep branch, close to the root of the archaeal tree. However, genome comparisons indicate that, in both trees constructed using concatenated alignments of ribosomal proteins and trees based on gene content, M. kandleri consistently groups with other archaeal methanogens. M. kandleri shares the set of genes implicated in methanogenesis and, in part, its operon organization with Methanococcus jannaschii and Methanothermobacter thermoautotrophicum. These findings indicate that archaeal methanogens are monophyletic. A distinctive feature of M. kandleri is the paucity of proteins involved in signaling and regulation of gene expression. Also, M. kandleri appears to have fewer genes acquired via lateral transfer than other archaea. These features might reflect the extreme habitat of this organism.