Use of the dTAG system in vivo to degrade CDK2 and CDK5 in adult mice and explore potential safety liabilities.

The degradation tag (dTAG) system for target protein degradation can remove proteins from biological systems without the drawbacks of some genetic methods, such as slow kinetics, lack of reversibility, low specificity, and the inability to titrate dosage. These drawbacks can make it difficult to compare toxicity resulting from genetic and pharmacological interventions, especially in vivo. Because the dTAG system has not been studied extensively in vivo, we explored the use of this system to study the physiological sequalae resulting from CDK2 or CDK5 degradation in adult mice. Mice with homozygous knock-in of the dTAG sequence onto CDK2 and CDK5 were born at Mendelian ratios despite decreased CDK2 or CDK5 protein levels in comparison with wild-type mice. In bone marrow cells and duodenum organoids derived from these mice, treatment with the dTAG degrader dTAG-13 resulted in rapid and robust protein degradation but caused no appreciable change in viability or the transcriptome. Repeated delivery of dTAG-13 in vivo for toxicity studies proved challenging; we explored multiple formulations in an effort to maximize degradation while minimizing formulation-related toxicity. Degradation of CDK2 or CDK5 in all organs except the brain, where dTAG-13 likely did not cross the blood brain barrier, only caused microscopic changes in the testis of CDK2dTAG mice. These findings were corroborated with conditional CDK2 knockout in adult mice. Our results suggest that the dTAG system can provide robust protein degradation in vivo and that loss of CDK2 or CDK5 in adult mice causes no previously unknown phenotypes.

Circulating microRNAs as promising testicular translatable safety biomarkers: current state and future perspectives.

Drug-induced testicular injury (DITI) is one of the often-observed and challenging safety issues seen during drug development. Semen analysis and circulating hormones currently utilized have significant gaps in their ability to detect testicular damage accurately. In addition, no biomarkers enable a mechanistic understanding of the damage to the different regions of the testis, such as seminiferous tubules, Sertoli, and Leydig cells. MicroRNAs (miRNAs) are a class of non-coding RNAs that modulate gene expression post-transcriptionally and have been indicated to regulate a wide range of biological pathways. Circulating miRNAs can be measured in the body fluids due to tissue-specific cell injury/damage or toxicant exposure. Therefore, these circulating miRNAs have become attractive and promising non-invasive biomarkers for assessing drug-induced testicular injury, with several reports on their use as safety biomarkers for monitoring testicular damage in preclinical species. Leveraging emerging tools such as 'organs-on-chips' that can emulate the human organ's physiological environment and function is starting to enable biomarker discovery, validation, and clinical translation for regulatory qualification and implementation in drug development.

Polyethlyene Glycol 200 Can Protect Rats Against Drug-Induced Kidney Toxicity Through Inhibition of the Renal Organic Anion Transporter 3.

MK-7680, a cyclic nucleotide prodrug, caused significant kidney tubule injury in female rats when administered orally at 1000 mg/kg/day for 2 weeks using 10% Polysorbate 80 as vehicle. However, kidney injury was absent when MK-7680 was administered at the same dose regimen using 100% Polyethylene Glycol 200 (PEG 200) as the vehicle. Subsequent investigations revealed that MK-7680 triphosphate concentrations in kidney were much lower in rats treated with MK-7680 using PEG 200 compared with 10% Polysorbate 80 vehicle, whereas plasma exposures of MK-7680 prodrug were similar. In vitro studies demonstrated that PEG 200 is an inhibitor of human renal uptake transporter organic anion transporter 3 (OAT3), of which MK-7680 is a substrate. Furthermore, PEG 200 and PEG 400 were found to interfere in vitro with human renal transporters OAT3, organic cation transporter (OCT) 2, multidrug resistance-associated protein (MRP) 2 and 4, and multidrug and toxin extrusion protein (MATE) 1 and 2K, but not OAT1. These results support a conclusion that PEG 200 may prevent MK-7680-induced kidney injury by inhibiting its active uptake into proximal tubular cells by OAT3. Caution should be exercised therefore when using PEGs as vehicles for toxicity assessment for compounds that are substrates of renal transporters.

Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice.

X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and is one of the most common causes of macular degeneration in young men. Our therapeutic approach for XLRS is based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal (IVT) route. Two Good Laboratory Practice studies, a 9-month study in New Zealand White rabbits (n = 124) injected with AAV8-scRS/IRBPhRS at doses of 2E9, 2E10, 2E11, and 1.5E12 vector genomes/eye (vg/eye), and a 6-month study in Rs1-KO mice (n = 162) dosed with 2E9 and 2E10 vg/eye of the same vector were conducted to assess ocular and systemic safety. A self-resolving, dose-dependent vitreal inflammation was the main ocular finding, and except for a single rabbit dosed with 1.5E12 vg/eye, which showed a retinal detachment, no other ocular adverse event was reported. Systemic toxicity was not identified in either species. Biodistribution analysis in Rs1-KO mice detected spread of vector genome in extraocular tissues, but no evidence of organ or tissues damage was found. These studies indicate that IVT administration of AAV8-scRS/IRBPhRS is safe and well tolerated and support its advancement into a phase 1/2a clinical trial for XLRS.

Repeat-dose toxicology evaluation in cynomolgus monkeys of AVI-4658, a phosphorodiamidate morpholino oligomer (PMO) drug for the treatment of duchenne muscular dystrophy.

AVI-4658 is a phosphorodiamidate morpholino oligomer (PMO) drug designed to restore dystrophin expression in a subset of patients with Duchenne muscular dystrophy (DMD). Previous reports demonstrated this clinical proof-of-principle in patients with DMD following intramuscular injection of AVI-4658. This preclinical study evaluated the toxicity and toxicokinetic profile of AVI-4658 when administered either intravenously (IV) or subcutaneously (SC) to cynomolgus monkeys once weekly over 12 weeks, at doses up to the maximum feasible dose of 320 mg/kg per injection. No drug-related effects were noted on survival, clinical observations, body weight, food consumption, opthalmoscopic or electrocardiographic evaluations, hematology, clinical chemistry, urinalysis, organ weights, and macroscopic evaluations. Drug-related microscopic renal effects were dose-dependent, apparently reversible, and included basophilic granules (minimal), basophilic tubules (minimal to moderate), and tubular vacuolation (minimal to mild). These data establish the tolerability of AVI-4658 at doses up to and including the maximum feasible dose of 320 mg/kg by IV bolus or SC injection.

Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni.

BACKGROUND: During gut colonization, the enteric pathogen Campylobacter jejuni must surmount the toxic effects of reactive oxygen species produced by its own metabolism, the host immune system, and intestinal microflora. Elucidation of C. jejuni oxidative stress defense mechanisms is critical for understanding Campylobacter pathophysiology. RESULTS: The mechanisms of oxidative stress defense in C. jejuni were characterized by transcriptional profiling and phenotypic analysis of wild-type and mutant strains. To define the regulon of the peroxide-sensing regulator, PerR, we constructed an isogenic DeltaperR mutant and compared its transcriptome profile with that of the wild-type strain. Transcriptome profiling identified 104 genes that belonged to the PerR regulon. PerR appears to regulate gene expression in a manner that both depends on and is independent of the presence of iron and/or H2O2. Mutation of perR significantly reduced motility. A phenotypic analysis using the chick colonization model showed that the DeltaperR mutant exhibited attenuated colonization behavior. An analysis of changes in the transcriptome induced by exposure to H2O2, cumene hydroperoxide, or menadione revealed differential expression of genes belonging to a variety of biological pathways, including classical oxidative stress defense systems, heat shock response, DNA repair and metabolism, fatty acid biosynthesis, and multidrug efflux pumps. Mutagenic and phenotypic studies of the superoxide dismutase SodB, the alkyl-hydroxyperoxidase AhpC, and the catalase KatA, revealed a role for these proteins in oxidative stress defense and chick gut colonization. CONCLUSION: This study reveals an interplay between PerR, Fur, iron metabolism and oxidative stress defense, and highlights the role of these elements in C. jejuni colonization of the chick cecum and/or subsequent survival.

Diffuse cutaneous mastocytosis in a newborn calf.

Previously reported cases of congenital cutaneous mast cell tumours in calves were characterized by multiple discrete nodules. The calf presented herein is, to the authors' knowledge, the first documented case of congenital diffuse cutaneous mastocytosis. The entire skin was thickened and wrinkled with particularly prominent skin folds on the head, ventrum, and legs. Histological examination of two haired skin specimens (eyelid, muzzle margin) revealed that the dermis was diffusely infiltrated by sheets of neoplastic mast cells.

Clinical and clinicopathologic features of dogs that consumed foodborne hepatotoxic aflatoxins: 72 cases (2005-2006).

OBJECTIVE: To characterize clinical signs, clinicopathologic features, treatments, and survival in dogs with naturally acquired foodborne aflatoxicosis. DESIGN: Retrospective case series. ANIMALS: 72 dogs that consumed aflatoxin-contaminated commercial dog food. PROCEDURES: Medical records of affected dogs were reviewed. Between December 2005 and March 2006, dogs were identified as having foodborne aflatoxin hepatotoxicosis on the basis of the history of consumption of contaminated food or characteristic histopathologic lesions (subject dog or a recently deceased dog in the same household or kennel). Recorded information included signalment, clinical features, clinicopathologic test results, treatments, and survival. Data were analyzed by survival status. RESULTS: Most dogs were of large breeds from breeding kennels. No significant differences were found in age or weight between 26 (36%) survivor dogs and 46 (64%) nonsurvivor dogs. Severity of clinical signs varied widely; 7 dogs died abruptly. In order of onset, clinical features included anorexia, lethargy, vomiting, jaundice, diarrhea (melena, hematochezia), abdominal effusion, peripheral edema, and terminal encephalopathy and hemorrhagic diathesis. Common clinicopathologic features included coagulopathic and electrolyte disturbances, hypoproteinemia, increased serum liver enzyme activities, hyperbilirubinemia, and hypocholesterolemia. Cytologic hepatocellular lipid vacuolation was confirmed in 11 dogs examined. In comparisons of clinicopathologic test results between survivor and nonsurvivor dogs, only granular cylindruria (7/21 dogs) consistently predicted death. Best early markers of aflatoxicosis were low plasma activities of anticoagulant proteins (protein C, antithrombin) and hypocholesterolemia. Despite aggressive treatment, many but not all severely affected dogs died. CONCLUSIONS AND CLINICAL RELEVANCE: Serum liver enzyme activities and bilirubin concentration were unreliable early markers of aflatoxin hepatotoxicosis in dogs. Hypocholesterolemia and decreased plasma protein C and antithrombin activities may function as exposure biomarkers.

Identification of Campylobacter jejuni genes contributing to acid adaptation by transcriptional profiling and genome-wide mutagenesis.

In order to cause disease, the food- and waterborne pathogen Campylobacter jejuni must face the extreme acidity of the host stomach as well as cope with pH fluctuations in the intestine. In the present study, C. jejuni NCTC 11168 was grown under mildly acidic conditions mimicking those encountered in the intestine. The resulting transcriptional profiles revealed how this bacterium fine-tunes gene expression in response to acid stress. This adaptation involves the differential expression of respiratory pathways, the induction of genes for phosphate transport, and the repression of energy generation and intermediary metabolism genes. We also generated and screened a transposon-based mutant library to identify genes required for wild-type levels of growth under mildly acidic conditions. This screen highlighted the important role played by cell surface components (flagella, the outer membrane, capsular polysaccharides, and lipooligosaccharides) in the acid stress response of C. jejuni. Our data also revealed that a limited correlation exists between genes required for growth under acidic conditions and genes differentially expressed in response to acid. To gain a comprehensive picture of the acid stress response of C. jejuni, we merged transcriptional profiles obtained from acid-adapted cells and cells subjected to acid shock. Genes encoding the transcriptional regulator PerR and putative oxidoreductase subunits Cj0414 and Cj0415 were among the few up-regulated under both acid stress conditions. As a Cj0415 mutant was acid sensitive, it is likely that these genes are crucial to the acid stress response of C. jejuni and consequently are important for host colonization.

Identification of Campylobacter jejuni genes involved in the response to acidic pH and stomach transit.

Campylobacter jejuni causes food- and waterborne gastroenteritis, and as such it must survive passage through the stomach in order to reach the gastrointestinal tract. While little is known about how C. jejuni survives transit through the stomach, its low infectious dose suggests it is well equipped to sense and respond to acid shock. In this study, the transcriptional profile of C. jejuni NCTC 11168 was obtained after the organism was exposed to in vitro and in vivo (piglet stomach) acid shock. The observed down-regulation of genes encoding ribosomal proteins likely reflects the need to reshuffle energy toward the expression of components required for survival. Acid shock also caused C. jejuni to up-regulate genes involved in stress responses. These included heat shock genes as well as genes involved in the response to oxidative and nitrosative stress. A role for the chaperone clpB in acid resistance was confirmed in vitro. Some genes showed expression patterns that were markedly different in vivo and in vitro, which likely reflects the complexity of the in vivo environment. For instance, transit through the stomach was characterized by up-regulation of genes that encode products that are involved in the use of nitrite as a terminal electron acceptor and down-regulation of genes that are involved in capsular polysaccharide expression. In conclusion, this study has enabled us to understand how C. jejuni modulates gene expression in response to acid shock in vitro and to correlate this with gene expression profiles of C. jejuni as it transits through the host stomach.

Major role for FeoB in Campylobacter jejuni ferrous iron acquisition, gut colonization, and intracellular survival.

To assess the importance of ferrous iron acquisition in Campylobacter physiology and pathogenesis, we disrupted and characterized the Fe2+ iron transporter, FeoB, in Campylobacter jejuni NCTC 11168, 81-176, and ATCC 43431. The feoB mutant was significantly affected in its ability to transport 55Fe2+. It accumulated half the amount of iron than the wild-type strain during growth in an iron-containing medium. The intracellular iron of the feoB mutant was localized in the periplasmic space versus the cytoplasm for the wild-type strain. These results indicate that the feoB gene of C. jejuni encodes a functional ferrous iron transport system. Reverse transcriptase PCR analysis revealed the cotranscription of feoB and Cj1397, which encodes a homolog of Escherichia coli feoA. C. jejuni 81-176 feoB mutants exhibited reduced ability to persist in human INT-407 embryonic intestinal cells and porcine IPEC-1 small intestinal epithelial cells compared to the wild type. C. jejuni NCTC 11168 feoB mutant was outcompeted by the wild type for colonization and/or survival in the rabbit ileal loop. The feoB mutants of the three C. jejuni strains were significantly affected in their ability to colonize the chick cecum. And finally, the three feoB mutants were outcompeted by their respective wild-type strains for infection of the intestinal tracts of colostrum-deprived piglets. Taken together, these results demonstrate that FeoB-mediated ferrous iron acquisition contributes significantly to colonization of the gastrointestinal tract during both commensal and infectious relationship, and thus it plays an important role in Campylobacter pathogenesis.

Use of genome-wide expression profiling and mutagenesis to study the intestinal lifestyle of Campylobacter jejuni.

Campylobacter jejuni is the most common bacterial cause of diarrhea worldwide. To colonize the gut and cause infection, C. jejuni must successfully compete with endogenous microbes for nutrients, resist host defenses, persist in the intestine, and ultimately infect the host. These challenges require the expression of a battery of colonization and virulence determinants. In this study, the intestinal lifestyle of C. jejuni was studied using whole-genome microarray, mutagenesis, and a rabbit ileal loop model. Genes associated with a wide range of metabolic, morphological, and pathological processes were expressed in vivo. The in vivo transcriptome of C. jejuni reflected its oxygen-limited, nutrient-poor, and hyperosmotic environment. Strikingly, the expression of several C. jejuni genes was found to be highly variable between individual rabbits. In particular, differential gene expression suggested that C. jejuni extensively remodels its envelope in vivo by differentially expressing its membrane proteins and by modifying its peptidoglycan and glycosylation composition. Furthermore, mutational analysis of seven genes, hspR, hrcA, spoT, Cj0571, Cj0178, Cj0341, and fliD, revealed an important role for the stringent and heat shock response in gut colonization. Overall, this study provides new insights on the mechanisms of gut colonization, as well as possible strategies employed by Campylobacter to resist or evade the host immune responses.

Iron acquisition and regulation in Campylobacter jejuni.

Iron affects the physiology of bacteria in two different ways: as a micronutrient for bacterial growth and as a catalyst for the formation of hydroxyl radicals. In this study, we used DNA microarrays to identify the C. jejuni genes that have their transcript abundance affected by iron availability. The transcript levels of 647 genes were affected after the addition of iron to iron-limited C. jejuni cells. Several classes of affected genes were revealed within 15 min, including immediate-early response genes as well as those specific to iron acquisition and metabolism. In contrast, only 208 genes were differentially expressed during steady-state experiments comparing iron-rich and iron-limited growth conditions. As expected, genes annotated as being involved in either iron acquisition or oxidative stress defense were downregulated during both time course and steady-state experiments, while genes encoding proteins involved in energy metabolism were upregulated. Because the level of protein glycosylation increased with iron limitation, iron may modulate the level of C. jejuni virulence by affecting the degree of protein glycosylation. Since iron homeostasis has been shown to be Fur regulated in C. jejuni, an isogenic fur mutant was used to define the Fur regulon by transcriptome profiling. A total of 53 genes were Fur regulated, including many genes not previously associated with Fur regulation. A putative Fur binding consensus sequence was identified in the promoter region of most iron-repressed and Fur-regulated genes. Interestingly, a fur mutant was found to be significantly affected in its ability to colonize the gastrointestinal tract of chicks, highlighting the importance of iron homeostasis in vivo. Directed mutagenesis of other genes identified by the microarray analyses allowed the characterization of the ferric enterobactin receptor, previously named CfrA. Chick colonization assays indicated that mutants defective in enterobactin-mediated iron acquisition were unable to colonize the gastrointestinal tract. In addition, a mutation in a receptor (Cj0178) for an uncharacterized iron source also resulted in reduced colonization potential. Overall, this work documents the complex response of C. jejuni to iron availability, describes the genetic network between the Fur and iron regulons, and provides insight regarding the role of iron in C. jejuni colonization in vivo.