Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

One size does not fit all: Caste and sex differences in the response of bumblebees (Bombus impatiens) to chronic oral neonicotinoid exposure.

Neonicotinoid insecticides have been implicated in the rapid global decline of bumblebees over recent years, particularly in agricultural and urban areas. While there is much known about neonicotinoid toxicity effects at the colony stage of the bumblebee annual cycle, far less is known about such effects at other stages critical for the maintenance of wild populations. In the present work, individual-based feeding assays were used to show that chronic consumption of the widely used neonicotinoid clothianidin at a field-realistic average rate of 3.6 and 4.0 ng/g·bee/day reduces survival of queen and male bumblebees, respectively, within a 7-day period. In contrast, worker survival was unaffected at a similar consumption rate of 3.9 ng/g·bee/day. To test the hypothesis that males have a lower tolerance for oral clothianidin exposure than workers due to their haploid genetic status, RNAseq analysis was used to compare the transcriptomic responses of workers and males to chronic intake of clothianidin at a sub-lethal dose of 0.37ng/bee/day for 5 days. Surprisingly, clothianidin consumption only altered the expression of 19 putative detoxification genes in a sex-specific manner, with 11/19 genes showing increased expression in workers. Sub-lethal clothianidin exposure also altered the expression of 40 genes associated with other major biological functions, including locomotion, reproduction, and immunity. Collectively, these results suggest that chronic oral toxicity effects of neonicotinoids are greatest during mating and nest establishment phases of the bumblebee life cycle. Chronic oral toxicity testing on males and queens is therefore required in order to fully assess the impact of neonicotinoids on wild bumblebee populations.

Immune-cognitive system connectivity reduces bumblebee foraging success in complex multisensory floral environments.

Bumblebees are declining at alarming rate worldwide, posing a significant threat to the function and diversity of temperate ecosystems. These declines have been attributed, in part, to the direct effect of specific pathogens on bumblebee survival. However, pathogens may also have a negative impact on host populations indirectly through immune-induced cognitive deficits in infected individuals. To gain greater insight into mechanisms and potential conservation implications of such 'immune-brain crosstalk' in bumblebees, we non-pathogenetically activated humoral and cellular immune pathways in individuals and then tested for long-term reductions in cognitive performance and foraging proficiency. We show that chronic activation of humoral, but not a cellular, immune pathways and effectors in foragers significantly reduces their ability to flexibly and efficiently harvest resources in multi-sensory floral environments for at least 7 days post-treatment. Humoral defense responses thus have the potential to confer significant foraging costs to bumblebee foragers over timeframes that would negatively impact colony growth and reproductive output under natural conditions. Our findings indicate that fitness effects of immune-brain crosstalk should be considered before attributing wild bumblebee decline to a particular pathogen species.

Helicobacter pylori infection does not promote hepatocellular cancer in a transgenic mouse model of hepatitis C virus pathogenesis.

Helicobacter pylori (H. pylori) and hepatitis C virus (HCV) infect millions of people and can induce cancer. We investigated if H. pylori infection promoted HCV-associated liver cancer. Helicobacter-free C3B6F1 wild-type (WT) and C3B6F1-Tg(Alb1-HCVN)35Sml (HT) male and female mice were orally inoculated with H. pylori SS1 or sterile media. Mice were euthanized at ~12 mo postinoculation and samples were collected for analyses. There were no significant differences in hepatocellular tumor promotion between WT and HT mice; however, HT female mice developed significantly larger livers with more hepatic steatosis than WT female mice. H. pylori did not colonize the liver nor promote hepatocellular tumors in WT or HT mice. In the stomach, H. pylori induced more corpus lesions in WT and HT female mice than in WT and HT male mice, respectively. The increased corpus pathology in WT and HT female mice was associated with decreased gastric H. pylori colonization, increased gastric and hepatic interferon gamma expression, and increased serum Th1 immune responses against H. pylori. HT male mice appeared to be protected from H. pylori-induced corpus lesions. Furthermore, during gastric H. pylori infection, HT male mice were protected from gastric antral lesions and hepatic steatosis relative to WT male mice and these effects were associated with increased serum TNF-α. Our findings indicate that H. pylori is a gastric pathogen that does not promote hepatocellular cancer and suggest that the HCV transgene is associated with amelioration of specific liver and gastric lesions observed during concurrent H. pylori infection in mice.

Helicobacter hepaticus--induced liver tumor promotion is associated with increased serum bile acid and a persistent microbial-induced immune response.

Chronic microbial infection influences cancer progression, but the mechanisms that link them remain unclear. Constitutive androstane receptor (CAR) is a nuclear receptor that regulates enzymes involved in endobiotic and xenobiotic metabolism. CAR activation is a mechanism of xenobiotic tumor promotion; however, the effects of chronic microbial infection on tumor promotion have not been studied in the context of CAR function. Here, we report that CAR limits the effects of chronic infection-associated progression of liver cancer. CAR knockout (KO) and wild-type (WT) male mice were treated with or without the tumor initiator diethylnitrosamine (DEN) at 5 weeks of age and then orally inoculated with Helicobacter hepaticus (Hh) or sterile media at 8 weeks of age. At approximately 50 weeks postinoculation, mice were euthanized for histopathologic, microbiological, molecular, and metabolomic analyses. Hh infection induced comparable hepatitis in WT and KO mice with or without DEN that correlated with significant upregulation of Tnfα and toll receptor Tlr2. Notably, DEN-treated Hh-infected KO mice exhibited increased numbers of liver lobes with dysplasia and neoplasia and increased multiplicity of neoplasia, relative to similarly treated WT mice. Enhanced tumor promotion was associated with decreased hepatic expression of P450 enzymes Cyp2b10 and Cyp3a11, increased expression of Camp, and increased serum concentrations of chenodeoxycholic acid. Together, our findings suggest that liver tumor promotion is enhanced by an impaired metabolic detoxification of endobiotics and a persistent microbial-induced immune response.

17ß-estradiol and tamoxifen prevent gastric cancer by modulating leukocyte recruitment and oncogenic pathways in Helicobacter pylori-infected INS-GAS male mice.

Helicobacter pylori infection promotes male predominant gastric adenocarcinoma in humans. Estrogens reduce gastric cancer risk and previous studies showed that prophylactic 17ß-estradiol (E2) in INS-GAS mice decreases H. pylori-induced carcinogenesis. We examined the effect of E2 and tamoxifen (TAM) on H. pylori-induced gastric cancer in male and female INS-GAS mice. After confirming robust gastric pathology at 16 weeks postinfection (WPI), mice were implanted with E2, TAM, both E2 and TAM, or placebo pellets for 12 weeks. At 28 WPI, gastric histopathology, gene expression, and immune cell infiltration were evaluated and serum inflammatory cytokines measured. After treatment, no gastric cancer was observed in H. pylori-infected males receiving E2 and/or TAM, whereas 40% of infected untreated males developed gastric cancer. E2, TAM, and their combination significantly reduced gastric precancerous lesions in infected males compared with infected untreated males (P < 0.001, 0.01, and 0.01, respectively). However, TAM did not alter female pathology regardless of infection status. Differentially expressed genes from males treated with E2 or TAM (n = 363 and n = 144, Q < 0.05) associated highly with cancer and cellular movement, indicating overlapping pathways in the reduction of gastric lesions. E2 or TAM deregulated genes associated with metastasis (PLAUR and MMP10) and Wnt inhibition (FZD6 and SFRP2). Compared with controls, E2 decreased gastric mRNA (Q < 0.05) and serum levels (P < 0.05) of CXCL1, a neutrophil chemokine, leading to decreased neutrophil infiltration (P < 0.01). Prevention of H. pylori-induced gastric cancer by E2 and TAM may be mediated by estrogen signaling and is associated with decreased CXCL1, decreased neutrophil counts, and downregulation of oncogenic pathways.