Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.

RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.

Low-level shRNA cytotoxicity can contribute to MYC-induced hepatocellular carcinoma in adult mice.

Short hairpin RNAs (shRNAs) have emerged as a novel therapeutic modality, but there is increasing concern over nonspecific effects in vivo. Here, we used viral vectors to express shRNAs against endogenous p53 in livers of conditional MYC-transgenic mice. As expected, the shRNAs silenced hepatic p53 and accelerated liver tumorigenesis when MYC was concurrently expressed. Surprisingly, various irrelevant control shRNAs similarly induced a rapid onset of tumorigenesis, comparable to carbon tetrachloride (CCl4), a potent carcinogen. We found that even marginal shRNA doses can already trigger histologically detectable hepatoxicity and increased hepatocyte apoptosis. Moreover, we noted that shRNA expression globally dysregulated hepatic microRNA (miRNA) expression, and that shRNA levels and activity further increased in the presence of MYC. In MYC-expressing transgenic mice, the marginal shRNA-induced liver injury sufficed to further stimulate hepatocellular division that was in turn associated with markedly increased expression of the mitotic cyclin B1. Hence, even at low doses, shRNAs can cause low-level hepatoxicity that can facilitate the ability of the MYC oncogene to induce liver tumorigenesis. Our data warrant caution regarding the possible carcinogenic potential of shRNAs when used as clinical agent, particularly in circumstances where tissues are genetically predisposed to cellular transformation and proliferation.

Neoplasia in 125 donkeys (Equus asinus): literature review and a survey of five veterinary schools in the United States and Canada.

A diagnosis of neoplasia was noted in 125 of 357 donkeys (35%) in our review of medical records from 5 veterinary schools in the United States and Canada. Equine sarcoid was the most common tumor in our study, accounting for 72% of all tumors and 82% of cutaneous tumors. Soft-tissue sarcomas were the second most common skin tumors. All other types of neoplasia were rare. Important differences in the occurrence of neoplasia in donkeys compared to horses included the rarity or absence of squamous cell carcinoma in any organ system and gray horse melanoma. Lymphosarcoma, the most common malignant tumor in horses, appears to be very rare in donkeys. We report several tumors in donkeys including melanocytoma, peripheral nerve sheath tumor, and gastrointestinal stromal tumor. Our data demonstrate commonalities as well as differences in neoplastic diseases of donkeys and horses. Understanding differences in carcinogenesis among these 2 closely related species can inform researchers pursuing pathogenic mechanisms of equine disease and inform veterinary diagnosticians regarding tumor prevalence.

Wnts as essential growth factors for the adult small intestine and colon.

The study of physiologic functions of Wnt proteins has been complicated by the redundant nature of the families encoding the Wnt factors and their Frizzled receptors. Adenoviral expression of the secreted Wnt antagonist Dickkopf-1 (Dkk1) was used to achieve fully conditional inhibition of canonical Wnt signaling in adult mice. Systemic expression of Dkk1 resulted in rapid inhibition of Wnt target gene expression and of proliferation of the small intestine and colon, loss of proliferative crypts, and eventual inflammation and architectural degeneration. These studies indicate an essential requirement for extracellular Wnt signaling in the maintenance of adult small intestine and colon proliferation. The essential role of Wnt signaling in ongoing proliferation in the colon suggests potential clinical applications in mucosal repair for inflammatory bowel diseases and underscores the utility of adenoviral strategies for conditional ablation of gene function in adult organisms.

Effects of husbandry practices, gender, and normal physiological variation on growth and reproduction of Japanese medaka, Oryzias latipes.

Japanese medaka, Oryzias latipes, are currently used in a variety of research applications for toxicological and carcinogenesis research, yet the impact of certain husbandry factors on study outcome has received limited attention. In this study, we demonstrated significant effects of stocking density (SD), dietary restriction (DR) and photoperiod on somatic growth in medaka. Higher stocking densities significantly inhibited somatic and hepatic growth in females, while having no similar effects on males. Daily egg production declined in a step-wise manner in groups of fish stocked at densities ranging from 0.38 to 2.6 fish per l. Significantly slower somatic growth was observed in fish fed on a mildly restrictive dietary protocol compared with those fish fed to excess. Further significant declines were observed in somatic growth of fish fed at a rate comparable to that used in research studies (3-4% body weight (BW) per day). Fish reared at an 8-h light:16-h dark (8L/16D) photoperiod were significantly smaller than cohorts reared at a 16-h light:8-h dark (16L/8D) photoperiod and female sexual maturity was delayed in the short photoperiod cohort. In normal female medaka, a high degree of individual variation was observed in daily egg production, as well as diurnal fluctuations in ovarian weight, gonadosomatic index (GSI) and hepatosomatic index (HSI). These results indicate some husbandry factors affect growth, sexual maturation and egg production in medaka and should be taken into consideration in studies evaluating endpoints that may be impacted by growth and reproductive performance, i.e. carcinogenesis and endocrine disruption studies. The high degree of individual variation among normal females and daily fluctuations in organ weights should also be considered in study design.

Modulation of peripheral B cell tolerance by CD72 in a murine model.

OBJECTIVE: B cells play a dominant role in the pathogenesis of several autoimmune diseases, including systemic lupus erythematosus. It is not well understood how B cell signaling contributes to autoantibody production. The goal of this study was to elucidate the role of CD72 in modulating B cell receptor (BCR)-mediated tolerogenic signaling and peripheral B cell tolerance. METHODS: A mouse model utilizing hen egg lysozyme (HEL) "anergic" B cells was studied. CD72-deficient mice carrying the BCR-specific IgHEL and/or soluble HEL (sHEL) transgenes were generated by breeding IgHEL-transgenic MD4 mice and/or sHEL-transgenic ML5 mice with congenic, CD72-deficient C57BL/6J mice. Normal and anergic B cells were isolated for analyses of B cell signaling. Aged wild-type and CD72-deficient mice were also examined for autoimmune phenomena. RESULTS: In the absence of CD72, anergic B cells inappropriately proliferated and survived in response to stimulation with self antigen. Biochemical analyses indicated that in anergic B cells, CD72 dominantly down-regulated BCR signaling to limit the antigen-induced elevation in [Ca2+]i and the activation of NFATc1, NF-kappaB, MAPK, and Akt. Mechanistically, CD72 was associated with, and regulated, the molecular adaptor Cbl-b in anergic B cells, suggesting that Cbl-b may play a role in mediating the negative effects of CD72 on BCR signaling. Moreover, in aged CD72-deficient mice, spontaneous production of antinuclear and anti-double-stranded DNA autoantibodies and features of lupus-like autoimmune disease were observed. CONCLUSION: CD72 is required to maintain B cell anergy and functions as a regulator of peripheral B cell tolerance. Thus, altered CD72 expression may play a role during the development of systemic lupus erythematosus.

A pilot toxicology study of single-walled carbon nanotubes in a small sample of mice.

Single-walled carbon nanotubes are currently under evaluation in biomedical applications, including in vivo delivery of drugs, proteins, peptides and nucleic acids (for gene transfer or gene silencing), in vivo tumour imaging and tumour targeting of single-walled carbon nanotubes as an anti-neoplastic treatment. However, concerns about the potential toxicity of single-walled carbon nanotubes have been raised. Here we examine the acute and chronic toxicity of functionalized single-walled carbon nanotubes when injected into the bloodstream of mice. Survival, clinical and laboratory parameters reveal no evidence of toxicity over 4 months. Upon killing, careful necropsy and tissue histology show age-related changes only. Histology and Raman microscopic mapping demonstrate that functionalized single-walled carbon nanotubes persisted within liver and spleen macrophages for 4 months without apparent toxicity. Although this is a preliminary study with a small group of animals, our results encourage further confirmation studies with larger groups of animals.

Naive and memory T cells induce different types of graft-versus-host disease.

The goal of this study was to compare the ability of donor naive and alloantigen-primed effector memory T cells to induce graft-vs-host disease after bone marrow transplantation in MHC-mismatched irradiated host mice. Purified CD4(+) naive (CD62L(high)CD44(low)) T cells and CD4(+) effector memory (CD62L(low)CD44(high)) T cells obtained from unprimed donors and donors primed to host alloantigens, respectively, were injected into host mice, and the rapidity, severity, and pattern of tissue injury of graft-vs-host disease was assessed. Unexpectedly, the naive T cells induced a more acute and severe colitis than the primed memory cells. Whereas the naive T cells expressing CD62L and CCR7 lymph node homing receptors vigorously expanded in mesenteric lymph nodes and colon by day 6 after transplantation, the primed memory T cells without these receptors had 20- to 100-fold lower accumulation at this early time point. These differences were reflected in the significantly more rapid decline in survival and weight loss induced by naive T cells. The primed memory T cells had a greater capacity to induce chronic colitis and liver injury and secrete IL-2 and IFN-gamma in response to alloantigenic stimulation compared with memory T cells from unprimed donors. Nevertheless, the expected increase in potency as compared with naive T cells was not observed due to differences in the pattern and kinetics of tissue injury.

Visual light spectroscopy reflects flow-related changes in brain oxygenation during regional low-flow perfusion and deep hypothermic circulatory arrest.

OBJECTIVES: Regional low-flow perfusion has been used to minimize ischemic brain injury during complex heart surgery in children. However, optimal regional low-flow perfusion remains undetermined. Visible light spectroscopy is a reliable method for continuous determination of capillary oxygen saturation (SgvO2). We used visible light spectroscopy to follow deep and superficial brain SgvO2 during cardiopulmonary bypass, regional low-flow perfusion, and deep hypothermic circulatory arrest. METHODS: Visible light spectroscopy probes were inserted into the superficial and deep brain of neonatal (3.9-4.5 kg) piglets, targeting the caudate and thalamic nuclei. The piglets were subjected to cardiopulmonary bypass and cooled to a rectal temperature of 18 degrees C using pH stat. Regional low-flow perfusion was initiated through the innominate artery at 18 degrees C, and pump flows were adjusted to 40, 30, 20, and 10 mL/kg/min for 10-minute intervals followed by 30 minutes of deep hypothermic circulatory arrest. Regional low-flow perfusion was reestablished, and flows were increased in a stepwise manner from 10 to 40 mL/kg/min. SgvO2 was continuously monitored. Carotid flow was measured using a flow probe, and cerebral blood flow (milliliters per kilogram body weight per minute) was calculated. RESULTS: There were no significant differences between the deep and superficial brain tissue oxygenation during regional low flow brain perfusion before deep hypothermic circulatory arrest. However, after deep hypothermic circulatory arrest, the superficial brain SgvO2 was lower than the deep brain SgvO2 (24 +/- 12 vs 55.3 +/- 8, P = .05, at flows of 30 mL/kg/min, and 34.2 +/- 17 vs 62.5 + 8, P = .06, at a flow rate of 40 mL/kg/min). During regional low-flow perfusion, SgvO2 was maintained at flows of 30 to 40 mL/kg/min (cerebral blood flows of 15 to 21 mL/kg/min and 19 to 24 mL/kg/min, respectively), but was significantly lower at pump flows of 20 mL/kg/min (cerebral blood flow of 10 to 14 mL/kg/min) and 10 mL/kg/min (cerebral blood flow of 5 to 9 mL/kg/min) compared with the values obtained just before regional low-flow perfusion (pre-deep hypothermic circulatory arrest, 37 +/- 6 vs 65.5 +/- 4.4, P < .05, and 21.6 +/- 3.7 vs 65.5 +/- 4.4, P < .01, respectively; and post-deep hypothermic circulatory arrest, 32 +/- 4.5 vs 65.5 +/- 4.4, P < .05, and 16.6 +/- 4.7 vs 65.5 +/- 4.4, P < .01, respectively). CONCLUSIONS: Regional low-flow perfusion at pump flows of 30 to 40 mL/kg/min with resulting cerebral blood flows of 14 to 24 mL/kg/min was adequate in maintaining both deep and superficial brain oxygenation. However, lower pump flows of 20 and 10 mL/kg/min, associated with cerebral blood flow of 9 to 14 mL/kg/min, resulted in significantly reduced SgvO2 values.

Antegrade cerebral perfusion reduces apoptotic neuronal injury in a neonatal piglet model of cardiopulmonary bypass.

OBJECTIVE: Neonates with congenital heart disease might require surgical repair with deep hypothermic circulatory arrest, a technique associated with adverse neurodevelopmental outcomes. Antegrade cerebral perfusion is thought to minimize ischemic brain injury, although there are no supporting experimental data. We sought to evaluate and compare the extent of neurologic injury in a neonatal piglet model of deep hypothermic circulatory arrest and antegrade cerebral perfusion. METHODS: Neonatal piglets undergoing cardiopulmonary bypass were randomized to deep hypothermic circulatory arrest or antegrade cerebral perfusion for 45 minutes. Animals were killed after 6 hours of recovery, and brain tissue was stained for evidence of cellular injury and for the apoptotic markers activated caspase 3 and cytochrome c translocation from mitochondria to cytosol. RESULTS: Piglets from the antegrade cerebral perfusion group exhibited less apoptotic or necrotic injury (4 +/- 3 vs 29 +/- 12 cells per field, P = .03). The piglets undergoing antegrade cerebral perfusion also had less evidence of apoptosis, with fewer cells staining for activated caspase 3 (57 +/- 8 vs 93 +/- 9 cells per field, P = .001) or showing cytochrome c translocation (6 +/- 2 vs 15 +/- 4 cells per field, P = .02). CONCLUSIONS: The use of antegrade cerebral perfusion in place of deep hypothermic circulatory arrest reduces evidence of apoptosis and histologic injury in neonatal piglets. Neonates with congenital heart disease might benefit from antegrade cerebral perfusion during complex cardiac surgery to improve their overall neurologic outcome.

Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1.

Whereas the adult gastrointestinal epithelium undergoes tremendous self-renewal through active proliferation in crypt stem cell compartments, the responsible growth factors regulating this continuous proliferation have not been defined. The exploration of physiologic functions of Wnt proteins in adult organisms has been hampered by functional redundancy and the necessity for conditional inactivation strategies. Dickkopf-1 (Dkk1) is a potent secreted Wnt antagonist that interacts with Wnt coreceptors of the LRP family. To address the contribution of Wnt signaling to gastrointestinal epithelial proliferation, adenoviral expression of Dkk1 was used to achieve stringent, conditional, and reversible Wnt inhibition in adult animals. Adenovirus Dkk1 (Ad Dkk1) treatment of adult mice repressed expression of the Wnt target genes CD44 and EphB2 within 2 days in both small intestine and colon, indicating an extremely broad role for Wnt signaling in the maintenance of adult gastrointestinal gene expression. In parallel, Ad Dkk1 markedly inhibited proliferation in small intestine and colon, accompanied by progressive architectural degeneration with the loss of crypts, villi, and glandular structure by 7 days. Whereas decreased Dkk1 expression at later time points (>10 days) was followed by crypt and villus regeneration, which was consistent with a reversible process, substantial mortality ensued from colitis and systemic infection. These results indicate the efficacy of systemic expression of secreted Wnt antagonists as a general strategy for conditional inactivation of Wnt signaling in adult organisms and illustrate a striking reliance on a single growth factor pathway for the maintenance of the architecture of the adult small intestine and colon.