Polymer-Mediated Inhibition of Pro-invasive Nucleic Acid DAMPs and Microvesicles Limits Pancreatic Cancer Metastasis.

Nucleic acid binding polymers (NABPs) have been extensively used as vehicles for DNA and RNA delivery. More recently, we discovered that a subset of these NABPs can also serve as anti-inflammatory agents by capturing pro-inflammatory extracellular nucleic acids and associated protein complexes that promote activation of toll-like receptors (TLRs) in diseases such as lupus erythematosus. Nucleic-acid-mediated TLR signaling also facilitates tumor progression and metastasis in several cancers, including pancreatic cancer (PC). In addition, extracellular DNA and RNA circulate on or within lipid microvesicles, such as microparticles or exosomes, which also promote metastasis by inducing pro-tumorigenic signaling in cancer cells and pre-conditioning secondary sites for metastatic establishment. Here, we explore the use of an NABP, the 3rd generation polyamidoamine dendrimer (PAMAM-G3), as an anti-metastatic agent. We show that PAMAM-G3 not only inhibits nucleic-acid-mediated activation of TLRs and invasion of PC tumor cells in vitro, but can also directly bind extracellular microvesicles to neutralize their pro-invasive effects as well. Moreover, we demonstrate that PAMAM-G3 dramatically reduces liver metastases in a syngeneic murine model of PC. Our findings identify a promising therapeutic application of NABPs for combating metastatic disease in PC and potentially other malignancies.

Utility of quantitative polymerase chain reaction in leptospirosis diagnosis: association of level of leptospiremia and clinical manifestations in Sri Lanka.

BACKGROUND: Quantitative polymerase chain reaction (qPCR), despite cost and logistical challenges, has the potential to provide accurate and timely diagnosis for leptospirosis at the point-of-care in endemic areas. We studied optimal sample types for qPCR, timing of sampling, and clinical manifestations in relation to quantitative leptospiremia. METHODS: A new qPCR assay using pathogenic Leptospira-specific 16S ribosomal RNA (rRNA) gene Taqman primers and an optimized temperature stepdown protocol was used to analyze patient blood samples. Serum was compared with whole blood as sample source. Quantitative leptospiremia was compared with clinical manifestations of leptospirosis and outcome. RESULTS: The diagnostic sensitivity of qPCR of whole blood and serum was 18.4% (95% confidence interval [CI]: 9.97%-31.4%) and 51.0% (95% CI: 37.5%-64.4%) respectively. The qPCR on suspected cases confirmed infection in 58 of 381 cases (15.2%). Of these, 6 cases confirmed by nested polymerase chain reaction (PCR) and sequencing were serologically negative using a standard but not regionally optimized microscopic agglutination test panel. The bacterial load in serum/blood ranged from 10(2) to 10(6) Leptospira/mL. Median leptospiral load for uncomplicated, renal failure, myocarditis, and multi-organ failure patients were 8616, 11007, 36100, and 15882 Leptospira/mL respectively. The qPCR window of positivity ranged from day 2 to day 15; sensitivity of qPCR was not affected by the length of the interval between the onset of symptoms and sample collection (P = .328). CONCLUSIONS: Quantitative PCR shows potential as a valid diagnostic test with a wider window of positivity than previously thought. Quantitative leptospiremia in serum/whole blood samples did not directly correlate with clinical manifestations of outcome in this patient population.

PEG-Like Brush Polymer Conjugate of RNA Aptamer That Shows Reversible Anticoagulant Activity and Minimal Immune Response.

Ribonucleic acid (RNA) therapeutics are an emerging class of drugs. RNA aptamers are of significant therapeutic and clinical interest because their activity can be easily reversed in vivo-a useful feature that is difficult to achieve using other therapeutic modalities. Despite their therapeutic promise, RNA aptamers are limited by their poor blood circulation. The attachment of polyethylene glycol (PEG) to RNA aptamers addresses this limitation. However, an RNA aptamer-PEG conjugate that is a reversible anticoagulant fails in a clinical trial due to the reactivity of the conjugate with pre-existing PEG antibodies and has cast a pall over PEGylation of aptamers and other biologics, despite its long history of utility in drug delivery. Here, PEG antibody-reactivity of this RNA aptamer is eliminated by conjugating it to a next-generation PEG-like brush polymer-poly[(oligoethylene glycol) methyl ether methacrylate)] (POEGMA). The conjugate retained the drug's therapeutic action and the ability to be easily reversed. Importantly, this conjugate does not bind pre-existing PEG antibodies that are prevalent in humans and does not induce a humoral immune response against the polymer itself in mice. These findings suggest a path to rescuing the PEGylation of RNA therapeutics and vaccines from the deleterious side-effects of PEG.

Anti-PEG Antibodies Inhibit the Anticoagulant Activity of PEGylated Aptamers.

Biopharmaceuticals have become increasingly attractive therapeutic agents and are often PEGylated to enhance their pharmacokinetics and reduce their immunogenicity. However, recent human clinical trials have demonstrated that administration of PEGylated compounds can evoke anti-PEG antibodies. Considering the ubiquity of PEG in commercial products and the presence of pre-existing anti-PEG antibodies in patients in large clinical trials evaluating a PEG-modified aptamer, we investigated how anti-PEG antibodies effect the therapeutic activities of PEGylated RNA aptamers. We demonstrate that anti-PEG antibodies can directly bind to and inhibit anticoagulant aptamer function in vitro and in vivo. Moreover, in parallel studies we detected the presence of anti-PEG antibodies in nonhuman primates after a single administration of a PEGylated aptamer. Our results suggest that anti-PEG antibodies can limit the activity of PEGylated drugs and potentially compromise the activity of otherwise effective therapeutic agents.

Nucleic acid scavenging microfiber mesh inhibits trauma-induced inflammation and thrombosis.

Trauma patients produce a host of danger signals and high levels of damage-associated molecular patterns (DAMPs) after cellular injury and tissue damage. These DAMPs are directly and indirectly involved in the pathogenesis of various inflammatory and thrombotic complications in patients with severe injuries. No effective therapeutic agents for the removal of DAMPs from blood or tissue fluid have been developed. Herein, we demonstrated that nucleic acid binding polymers, e.g., polyethylenimine (PEI) and polyamidoamine dendrimers, immobilized onto electrospun microfiber mesh can effectively capture various DAMPs, such as extracellular DNAs and high mobility group box 1 (HMGB1). Furthermore, treatment with PEI-immobilized microfiber mesh abrogated the ability of DAMPs, released from dead and dying cells in culture or found in patients following traumatic injury, to activate innate immune responses and coagulation in vitro and in vivo. Nucleic acid scavenging microfiber meshes represent an effective strategy to combat inflammation and thrombosis in trauma.

Utility and limitations of direct multi-locus sequence typing on qPCR-positive blood to determine infecting Leptospira strain.

Culture-independent molecular characterization of infecting Leptospira human blood specimens from a 2008 outbreak of human leptospirosis in central Sri Lanka was carried out. Of 58 quantitative real-time polymerase chain reaction-positive samples analyzed for seven multi-locus sequence typing (MLST) housekeeping genes (mreA, pfkB, pntA, sucA, tpiA, fadD, and glmU), interpretable data was obtained from 12 samples. Mean bacterial load was 2.2 × 10(5) among specimens with complete MLST profiles compared with 1.3 × 10(4) among specimens without complete MLST profiles; all specimens with complete profiles had at least 4.9 × 10(4) Leptospira/mL (t = 5, P < 0.001). Most (11/12) identified sequence types were ST1 (L. interrogans serovar Lai) and ST44 (L. interrogans serovar Geyaweera). MLST can be used to directly identify infecting Leptospira strains in blood samples obtained during acute illness without the need for culture isolation, but it shows important limitations related to bacterial load.

Pathogenomic inference of virulence-associated genes in Leptospira interrogans.

Leptospirosis is a globally important, neglected zoonotic infection caused by spirochetes of the genus Leptospira. Since genetic transformation remains technically limited for pathogenic Leptospira, a systems biology pathogenomic approach was used to infer leptospiral virulence genes by whole genome comparison of culture-attenuated Leptospira interrogans serovar Lai with its virulent, isogenic parent. Among the 11 pathogen-specific protein-coding genes in which non-synonymous mutations were found, a putative soluble adenylate cyclase with host cell cAMP-elevating activity, and two members of a previously unstudied ∼15 member paralogous gene family of unknown function were identified. This gene family was also uniquely found in the alpha-proteobacteria Bartonella bacilliformis and Bartonella australis that are geographically restricted to the Andes and Australia, respectively. How the pathogenic Leptospira and these two Bartonella species came to share this expanded gene family remains an evolutionary mystery. In vivo expression analyses demonstrated up-regulation of 10/11 Leptospira genes identified in the attenuation screen, and profound in vivo, tissue-specific up-regulation by members of the paralogous gene family, suggesting a direct role in virulence and host-pathogen interactions. The pathogenomic experimental design here is generalizable as a functional systems biology approach to studying bacterial pathogenesis and virulence and should encourage similar experimental studies of other pathogens.

Whole genome analysis of Leptospira licerasiae provides insight into leptospiral evolution and pathogenicity.

The whole genome analysis of two strains of the first intermediately pathogenic leptospiral species to be sequenced (Leptospira licerasiae strains VAR010 and MMD0835) provides insight into their pathogenic potential and deepens our understanding of leptospiral evolution. Comparative analysis of eight leptospiral genomes shows the existence of a core leptospiral genome comprising 1547 genes and 452 conserved genes restricted to infectious species (including L. licerasiae) that are likely to be pathogenicity-related. Comparisons of the functional content of the genomes suggests that L. licerasiae retains several proteins related to nitrogen, amino acid and carbohydrate metabolism which might help to explain why these Leptospira grow well in artificial media compared with pathogenic species. L. licerasiae strains VAR010(T) and MMD0835 possess two prophage elements. While one element is circular and shares homology with LE1 of L. biflexa, the second is cryptic and homologous to a previously identified but unnamed region in L. interrogans serovars Copenhageni and Lai. We also report a unique O-antigen locus in L. licerasiae comprised of a 6-gene cluster that is unexpectedly short compared with L. interrogans in which analogous regions may include >90 such genes. Sequence homology searches suggest that these genes were acquired by lateral gene transfer (LGT). Furthermore, seven putative genomic islands ranging in size from 5 to 36 kb are present also suggestive of antecedent LGT. How Leptospira become naturally competent remains to be determined, but considering the phylogenetic origins of the genes comprising the O-antigen cluster and other putative laterally transferred genes, L. licerasiae must be able to exchange genetic material with non-invasive environmental bacteria. The data presented here demonstrate that L. licerasiae is genetically more closely related to pathogenic than to saprophytic Leptospira and provide insight into the genomic bases for its infectiousness and its unique antigenic characteristics.