Longitudinal Neuropathological Consequences of Extracranial Radiation Therapy in Mice.

Cancer-related cognitive impairment (CRCI) is a consequence of chemotherapy and extracranial radiation therapy (ECRT). Our prior work demonstrated gliosis in the brain following ECRT in SKH1 mice. The signals that induce gliosis were unclear. Right hindlimb skin from SKH1 mice was treated with 20 Gy or 30 Gy to induce subclinical or clinical dermatitis, respectively. Mice were euthanized at 6 h, 24 h, 5 days, 12 days, and 25 days post irradiation, and the brain, thoracic spinal cord, and skin were collected. The brains were harvested for spatial proteomics, immunohistochemistry, Nanostring nCounter® glial profiling, and neuroinflammation gene panels. The thoracic spinal cords were evaluated by immunohistochemistry. Radiation injury to the skin was evaluated by histology. The genes associated with neurotransmission, glial cell activation, innate immune signaling, cell signal transduction, and cancer were differentially expressed in the brains from mice treated with ECRT compared to the controls. Dose-dependent increases in neuroinflammatory-associated and neurodegenerative-disease-associated proteins were measured in the brains from ECRT-treated mice. Histologic changes in the ECRT-treated mice included acute dermatitis within the irradiated skin of the hindlimb and astrocyte activation within the thoracic spinal cord. Collectively, these findings highlight indirect neuronal transmission and glial cell activation in the pathogenesis of ECRT-related CRCI, providing possible signaling pathways for mitigation strategies.

Inhibition of lung adenocarcinoma by combinations of sulfasalazine (SAS) and disulfiram-copper (DSF-Cu) in cell line models and mice.

Sulfasalazine (SAS) is a repurposed antitumor drug which inhibits the proliferation and survival of cancer cells by inhibiting the xCT cellular antioxidant system. Recent clinical studies have shown that, due to poor bioavailability, the antitumor effects of SAS monotherapy are minimal. Therefore, we hypothesized that DSF, another repurposed drug that has demonstrated anticancer effects, or its complex with copper (DSF-copper, DSF-Cu) could potentiate the antilung cancer effects of SAS. Exposure of non-small cell lung cancer cells to therapeutically achievable concentrations of SAS-induced low-to-moderate cytotoxic effects (20-40% reduction in cell viability) and, unexpectedly, induced the antioxidant protein NRF2 and its downstream effectors xCT and ALDH1A1. However, combinations of SAS and DSF-Cu, but not SAS and DSF, induced a significantly higher cytotoxic effect (64-88% reduction in cell viability), apoptosis and generation of mitochondrial reactive oxygen species as compared with SAS or DSF-Cu alone. Moreover, DSF-Cu abrogated SAS-induced NRF2, xCT and ALDH1A1 expression. In a mouse model of lung tumor, SAS + DSF-Cu showed a higher efficacy than the individual drugs in reducing the number and size of tumors as well as the incidence and multiplicity of lung adenocarcinoma. Taken together, our findings indicate that the observed antilung cancer effects of SAS plus DSF-Cu are mediated, at least in part, via impairment of reactive oxygen species defense and -enhancement of oxidative stress and provide evidence for the preventive/therapeutic potential of this combinatorial approach against lung cancer.

Generation and characterization of an immunodeficient mouse model of mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (Hunter syndrome, MPS II) is an inherited X-linked recessive disease caused by deficiency of iduronate-2-sulfatase (IDS), resulting in the accumulation of the glycosaminoglycans (GAG) heparan and dermatan sulfates. Mouse models of MPS II have been used in several reports to study disease pathology and to conduct preclinical studies for current and next generation therapies. Here, we report the generation and characterization of an immunodeficient mouse model of MPS II, where CRISPR/Cas9 was employed to knock out a portion of the murine IDS gene on the NOD/SCID/Il2rγ (NSG) immunodeficient background. IDS-/- NSG mice lacked detectable IDS activity in plasma and all analyzed tissues and exhibited elevated levels of GAGs in those same tissues and in the urine. Histopathology revealed vacuolized cells in both the periphery and CNS of NSG-MPS II mice. This model recapitulates skeletal disease manifestations, such as increased zygomatic arch diameter and decreased femur length. Neurocognitive deficits in spatial memory and learning were also observed in the NSG-MPS II model. We anticipate that this new immunodeficient model will be appropriate for preclinical studies involving xenotransplantation of human cell products intended for the treatment of MPS II.

Combinatory lung tumor inhibition by myo-inositol and iloprost/rapamycin: association with immunomodulation.

Although both preclinical and clinical studies have suggested that myo-inositol (MI) may be a safe and effective lung cancer chemopreventive agent, its efficacy is moderate. To test whether the chemopreventive agents iloprost (IL) or rapamycin enhance the lung tumor inhibitory effects of MI, A/J mice were treated with the tobacco smoke carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and, beginning one week after the end of NNK treatment, given MI, IL, rapamycin, MI + IL or MI + rapamycin for 17 weeks. Analyses of the number and size of tumors on the surface of the lung have indicated that MI, IL, rapamycin, MI + IL and MI + rapamycin reduced the multiplicity of NNK-induced lung tumors by 41, 34, 46, 79 and 67%, respectively, and larger tumors (lung tumors with a diameter of 1-2 or >2 mm) were absent in the MI + IL and MI + rapamycin groups. These results clearly indicated that MI + IL and MI + rapamycin are more effective than MI alone in inhibiting the formation and growth of lung tumors. Assessment of the immunomodulatory effects of the drugs showed that whereas MI + rapamycin and MI + IL increased the infiltration of lung tumors by CD4+ and CD8+ T cells, MI + rapamycin reduced the expression of the immune checkpoint protein programmed-death ligand-1 (PD-L1). Moreover, all treatments, except IL, increased apoptosis, whereas cell proliferation was markedly suppressed in all treated groups. In summary, these results suggest that IL and rapamycin could enhance the efficacy of MI in lung cancer chemoprevention trials.

Contribution of the innate and adaptive immune systems to aortic dilation in murine mucopolysaccharidosis type I.

BACKGROUND: Adult immunocompetent male C57Bl/6 mucopolysaccharidosis, type I (MPSI) mice develop aortic insufficiency (AI), dilated ascending aortas and decreased cardiac function, findings not observed in immune incompetent adult male NSG MPSI mice. We sought to determine why. METHODS: Cardiac ultrasound measurements of ascending aorta and left ventricular dimensions and Doppler interrogation for AI were performed in 6-month-old male B6 MPSI (N = 12), WT (N = 6), NSG MPSI (N = 8), NSG (N = 6) mice. Urinary glycosaminoglycans, RNA sequencing with quantitative PCR were performed and aortic pathology assessed by routine and immunohistochemical staining on subsets of murine aortas. RESULTS: Ascending aortic diameters were significantly greater, left ventricular function significantly decreased, and AI significantly more frequent in B6 MPSI mice compared to NSG MPSI mice (p < 0.0001, p = 0.008 and p = 0.02, respectively); NSG and B6 WT mice showed no changes. Urinary glycosaminoglycans were significantly greater in B6 and NSG MPSI mice and both were significantly elevated compared to WT controls (p = 0.003 and p < 0.0001, respectively). By RNA sequencing, all 11 components of the inflammasome pathway were upregulated in B6 MUT, but only Aim2 and Ctsb in NSG MUT mice and none in WT controls. Both B6 and NSG MUT mice demonstrated variably-severe intramural inflammation, vacuolated cells, elastin fragmentation and disarray, and intense glycosaminoglycans on histological staining. B6 MPSI mice demonstrated numerous medial MAC2+ macrophages and adventitial CD3+ T-cells while MAC2+ macrophages were sparse and CD3+ T-cells absent in NSG MPSI mice. CONCLUSIONS: Aortic dilation, AI and decreased cardiac function occur in immunocompetent B6 MPSI male mice but not in immune incompetent NSG MPSI mice, unrelated to GAG excretion, upregulation of Ctsb, or routine histologic appearance. Upregulation of all components of the inflammasome pathway in B6 MUT, but not NSG MUT mice, and abundant medial MAC2 and adventitial CD3 infiltrates in B6, but not NSG, MPSI aortas differentiated the two strains. These results suggest that the innate and adaptive immune systems play a role in these cardiac findings which may be relevant to human MPSI.

Acrolein Increases the Pulmonary Tumorigenic Activity of the Tobacco-Specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

Tobacco smoke is a complex mixture of more than 7000 chemicals, of which many are toxic and/or carcinogenic. Many hazard assessments of tobacco have focused on individual chemical exposures without consideration of how the chemicals may interact with one another. Two chemicals, the human carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) and a possible human carcinogen, acrolein, were hypothesized to interact with one another, possibly owing to the additive effects of DNA adduct formation or influence on the repair of mutagenic DNA adducts. To test our hypothesis that coexposure to NNK and acrolein is more carcinogenic than either chemical alone, A/J mice were exposed to NNK (i.p., 0, 2.5, or 7.5 µmol in saline) in the presence or absence of inhaled acrolein (15 ppmV). While the single 3 h exposure to acrolein alone did not induce lung adenomas, it significantly enhanced NNK's lung carcinogenicity. In addition, mice receiving both NNK and acrolein had more adenomas with dysplasia or progression than those receiving only NNK, suggesting that acrolein may also increase the severity of NNK-induced lung adenomas. To test the hypothesis that the interaction was due to effects on DNA adduct formation and repair, NNK- and acrolein pulmonary DNA adduct levels were assessed. There was no consistent effect of the coexposure on NNK-derived DNA adducts, and acrolein DNA adducts were not elevated above endogenous levels. This study supports the hypothesis that tobacco smoke chemicals combine to contribute to the carcinogenic potency of tobacco smoke, and the mechanism of interaction cannot be explained by alterations of DNA adduct levels.

Juvenile exposure to doxorubicin alters the cardiovascular response to adult-onset psychosocial stress in mice.

Childhood cancer survivors have a high risk for premature cardiovascular diseases, mainly due to cardiotoxic cancer treatments such as doxorubicin (DOX). Psychosocial stress is a significant cardiovascular risk factor and an enormous burden in childhood cancer survivors. Although observational studies suggest that psychosocial stress is associated with cardiovascular complications in cancer survivors, there is no translationally relevant animal model to study this interaction. We established a "two-hit" model in which juvenile mice were administered DOX (4 mg/kg/week for 3 weeks), paired to a validated model of chronic subordination stress (CSS) 5 weeks later upon reaching adulthood. Blood pressure, heart rate, and activity were monitored by radio-telemetry. At the end of CSS experiment, cardiac function was assessed by echocardiography. Cardiac fibrosis and inflammation were assessed by histopathologic analysis. Gene expressions of inflammatory and fibrotic markers were determined by PCR. Juvenile exposure to DOX followed by adult-onset CSS caused cardiac fibrosis and inflammation as evident by histopathologic findings and upregulated gene expression of multiple inflammatory and fibrotic markers. Intriguingly, juvenile exposure to DOX blunted CSS-induced hypertension but not CSS-induced tachycardia. There were no significant differences in cardiac function parameters among all groups, but juvenile exposure to DOX abrogated the hypertrophic response to CSS. In conclusion, we established a translationally relevant mouse model of juvenile DOX-induced cardiotoxicity that predisposes to adult-onset stress-induced adverse cardiac remodeling. Psychosocial stress should be taken into consideration in cardiovascular risk stratification of DOX-treated childhood cancer survivors.

Reprogramming responsiveness to checkpoint blockade in dysfunctional CD8 T cells.

Established T cell dysfunction is a barrier to antitumor responses, and checkpoint blockade presumably reverses this. Many patients fail to respond to treatment and/or develop autoimmune adverse events. The underlying reason for T cell responsiveness remains elusive. Here, we show that susceptibility to checkpoint blockade is dependent on the activation status of T cells. Newly activated self-specific CD8 T cells respond to checkpoint blockade and cause autoimmunity, which is mitigated by inhibiting the mechanistic target of rapamycin. However, once tolerance is established, self-specific CD8 T cells display a gene signature comparable to tumor-specific CD8 T cells in a fixed state of dysfunction. Tolerant self-specific CD8 T cells do not respond to single or combinatorial dosing of anti-CTLA4, anti-PD-L1, anti-PD-1, anti-LAG-3, and/or anti-TIM-3. Despite this, T cell responsiveness can be induced by vaccination with cognate antigen, which alters the previously fixed transcriptional signature and increases antigen-sensing machinery. Antigenic reeducation of tolerant T cells synergizes with checkpoint blockade to generate functional CD8 T cells, which eliminate tumors without concomitant autoimmunity and are transcriptionally distinct from classic effector T cells. These data demonstrate that responses to checkpoint blockade are dependent on the activation state of a T cell and show that checkpoint blockade-insensitive CD8 T cells can be induced to respond to checkpoint blockade with robust antigenic stimulation to participate in tumor control.

Zygomatic sialocele secondary to infarction treated with sialoadenectomy in a dog.

Zygomatic salivary gland disease is not commonly reported in dogs and there is a paucity of literature reporting salivary gland disease secondary to infarction in dogs. A 9-year-old German wirehaired pointer presented with left eye exophthalmos, 3rd eyelid elevation, negative retropulsion, and pain upon opening of the mouth. Computed tomography revealed a mass extending from the left zygomatic salivary gland, consistent with a sialocele. A left-sided zygomatic sialoadenectomy was performed successfully. Histopathologic diagnosis concluded zygomatic salivary gland infarction. The dog had no signs of recurrence 20 mo after surgery. Key clinical message: To the authors' knowledge, this is the first case report with long-term outcome of a zygomatic sialocele secondary to salivary gland infarction in a dog treated by zygomatic sialoadenectomy via zygomatic osteotomy.

Sialocèle zygomatique secondaire à un infarctus traité par sialoadénectomie chez un chien. Une atteinte des glandes salivaires zygomatiques n'est pas fréquemment rapportée chez le chien et il existe peu de littérature rapportant une maladie des glandes salivaires secondaire à un infarctus chez le chien. Un pointeur allemand à poil dur de 9 ans présentait une exophtalmie de l'oeil gauche, une élévation de la troisième paupière, une rétropulsion négative et une douleur à l'ouverture de la bouche. La tomodensitométrie a révélé une masse s'étendant à partir de la glande salivaire zygomatique gauche, compatible avec une sialocèle. Une sialoadénectomie zygomatique gauche a été réalisée avec succès. Le diagnostic histopathologique a permis de conclure à un infarctus des glandes salivaires zygomatiques. Le chien n'avait aucun signe de récidive 20 mois après la chirurgie.Message clinique clé :À la connaissance des auteurs, il s'agit du premier rapport de cas avec évolution à long terme d'une sialocèle zygomatique secondaire à un infarctus des glandes salivaires chez un chien traité par sialoadénectomie zygomatique via une ostéotomie zygomatique.(Traduit par Dr Serge Messier).

Calibrated Bioresorbable Microspheres as an Embolic Agent: An Experimental Study in a Rabbit Renal Model.

PURPOSE: To evaluate the time frame of resorption and tissue response of newly developed bioresorbable microspheres (BRMS) and vessel recanalization after renal embolization. MATERIALS AND METHODS: Embolization of lower poles of kidneys of 20 adult rabbits was performed with BRMS (300-500 µm). Two rabbits were sacrificed immediately after embolization (day 0). Three rabbits were sacrificed after follow-up angiography at 3, 7, 10, 14, 21, and 30 days. The pathologic changes in the renal parenchyma, BRMS degradation, and vessel recanalization were evaluated histologically and angiographically. RESULTS: Embolization procedures were successfully performed, and all animals survived without complication. Infarcts were observed in all kidneys that received embolization harvested after day 0. Moderate degradation of BRMS (score = 1.07 ± 0.06) was observed by day 3. Of BRMS, 95% were resorbed before day 10 with scant BRMS materials remaining in the arteries at later time points. Partial vessel recanalization was observed by angiography starting on day 3, whereas new capillary formation was first identified histologically on day 7. Vascular inflammation associated with BRMS consisted of acute, heterophilic infiltrate at earlier time points (day 3 to day 10); this was resolved with the resorption of BRMS. Inflammation and fibrosis within infarcted regions were consistent with progression of infarction. CONCLUSIONS: BRMS were bioresorbable in vivo, and most BRMS were resorbed before day 10 with a mild tissue reaction. Vessel recanalization occurred secondary to the resorption of BRMS.

Susceptibility of domestic cats to chronic wasting disease.

Domestic and nondomestic cats have been shown to be susceptible to feline spongiform encephalopathy (FSE), almost certainly caused by consumption of bovine spongiform encephalopathy (BSE)-contaminated meat. Because domestic and free-ranging nondomestic felids scavenge cervid carcasses, including those in areas affected by chronic wasting disease (CWD), we evaluated the susceptibility of the domestic cat (Felis catus) to CWD infection experimentally. Cohorts of 5 cats each were inoculated intracerebrally (i.c.) or orally (p.o.) with CWD-infected deer brain. At 40 and 42 months postinoculation, two i.c.-inoculated cats developed signs consistent with prion disease, including a stilted gait, weight loss, anorexia, polydipsia, patterned motor behaviors, head and tail tremors, and ataxia, and the cats progressed to terminal disease within 5 months. Brains from these two cats were pooled and inoculated into cohorts of cats by the i.c., p.o., and intraperitoneal and subcutaneous (i.p./s.c.) routes. Upon subpassage, feline CWD was transmitted to all i.c.-inoculated cats with a decreased incubation period of 23 to 27 months. Feline-adapted CWD (Fel(CWD)) was demonstrated in the brains of all of the affected cats by Western blotting and immunohistochemical analysis. Magnetic resonance imaging revealed abnormalities in clinically ill cats, which included multifocal T2 fluid attenuated inversion recovery (FLAIR) signal hyperintensities, ventricular size increases, prominent sulci, and white matter tract cavitation. Currently, 3 of 4 i.p./s.c.- and 2 of 4 p.o. secondary passage-inoculated cats have developed abnormal behavior patterns consistent with the early stage of feline CWD. These results demonstrate that CWD can be transmitted and adapted to the domestic cat, thus raising the issue of potential cervid-to-feline transmission in nature.

Aerosol transmission of chronic wasting disease in white-tailed deer.

While the facile transmission of chronic wasting disease (CWD) remains incompletely elucidated, studies in rodents suggest that exposure of the respiratory mucosa may be an efficient pathway. The present study was designed to address this question in the native cervid host. Here, we demonstrate aerosol transmission of CWD to deer with a prion dose >20-fold lower than that used in previous oral inoculations. Inhalation of prions may facilitate transmission of CWD and, perhaps, other prion infections.

Genetic depletion of complement receptors CD21/35 prevents terminal prion disease in a mouse model of chronic wasting disease.

The complement system has been shown to facilitate peripheral prion pathogenesis. Mice lacking complement receptors CD21/35 partially resist terminal prion disease when infected i.p. with mouse-adapted scrapie prions. Chronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, similar to scrapie, has been shown to involve the immune system, which probably contributes to their relatively facile horizontal and environmental transmission. In this study, we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. CD21/35-deficient Tg5037 mice exhibit greatly impaired splenic prion accumulation and replication throughout disease, similar to CD21/35-deficient murine prion protein mice infected with mouse scrapie. TgA5037;CD21/35(-/-) mice exhibited little or no neuropathology and deposition of misfolded, protease-resistant prion protein associated with CWD. CD21/35 translocate to lipid rafts and mediates a strong germinal center response to prion infection that we propose provides the optimal environment for prion accumulation and replication. We further propose a potential role for CD21/35 in selecting prion quasi-species present in prion strains that may exhibit differential zoonotic potential compared with the parental strains.

Cancer related cognitive impairment: a downside of cancer treatment.

Cancer treatment is associated with long lasting cognitive impairment in cancer survivors. This cognitive impairment is often termed cancer related cognitive impairment (CRCI). Cancer survivors treated for tumors outside the central nervous system are increasingly diagnosed with CRCI. The development of strategies to mitigate the negative effects of cancer treatment on the brain are crucial. Although neuroimaging research has proposed several candidate mechanisms, the pathogenic underpinnings of CRCI remain uncertain. As such, preventative and treatment strategies have not been identified. To fill these gaps, animal models play a vital role in isolating underlying contributing mechanisms that promote CRCI and in testing new therapeutic approaches.

Deletion of Glyoxalase 1 Exacerbates Acetaminophen-Induced Hepatotoxicity in Mice.

Acetaminophen (APAP) overdose triggers a cascade of intracellular oxidative stress events, culminating in acute liver injury. The clinically used antidote, N-acetylcysteine (NAC), has a narrow therapeutic window, and early treatment is essential for a satisfactory therapeutic outcome. For more versatile therapies that can be effective even at late presentation, the intricacies of APAP-induced hepatotoxicity must be better understood. Accumulation of advanced glycation end products (AGEs) and the consequent activation of the receptor for AGEs (RAGE) are considered one of the key mechanistic features of APAP toxicity. Glyoxalase 1 (Glo-1) regulates AGE formation by limiting the levels of methylglyoxal (MEG). In this study, we studied the relevance of Glo-1 in the APAP-mediated activation of RAGE and downstream cell death cascades. Constitutive Glo-1-knockout mice (GKO) and a cofactor of Glo-1, ψ-GSH, were used as tools. Our findings showed elevated oxidative stress resulting from the activation of RAGE and hepatocyte necrosis through steatosis in GKO mice treated with high-dose APAP compared to wild-type controls. A unique feature of the hepatic necrosis in GKO mice was the appearance of microvesicular steatosis as a result of centrilobular necrosis, rather than the inflammation seen in the wild type. The GSH surrogate and general antioxidant ψ-GSH alleviated APAP toxicity irrespective of the Glo-1 status, suggesting that oxidative stress is the primary driver of APAP toxicity. Overall, the exacerbation of APAP hepatotoxicity in GKO mice suggests the importance of this enzyme system in antioxidant defense against the initial stages of APAP overdose.

To Compare the Effects of a Standard Versus Hydrophilic Polymer Coated Airway Stent in a Porcine Model: A Randomized, Single-Blinded Study.

BACKGROUND: Stent encrustation with debris and mucostasis is a significant cause of airway injury and comorbidity, leading to ~25% of stent exchanges (1-3). Previous work from our group has shown that the experimental coating can reduce mucous adhesion in bench testing and demonstrated a signal for reducing airway injury and mucostasis in a feasibility study. OBJECTIVES: The aim of this study is to continue our inquiry in a randomized, single-blinded multi-animal trial to investigate the degree of airway injury and mucostasis using silicone stents with and without this specialized coating. METHODS: We modified commercially available silicone stents with a hydrophilic polymer from Toray Industries. We conducted an in vivo survival study in 6 mainstem airways (3 coated and 3 uncoated) of 3 pigs to compare the degree of airway injury and mucostasis between coated versus noncoated stented airways. Both stents were randomized to either left or right mainstem bronchus. The pathologist was blinded to the stent type. RESULTS: We implanted a total of six 14×15 mm silicone stents (1 per mainstem bronchi) into 3 pigs. All animals survived to termination at 4 weeks. All stents were intact; however, 1 uncoated stent migrated out. On average, all the coated stents demonstrated reduced pathology and tissue injury scores (75 vs. 68.3, respectively). The average total dried mucous weight was slightly higher in the coated stents (0.07 g vs. 0.05 g; respectively). CONCLUSION: Coated stents had lower airway injury compared with uncoated stents in this study. Of all the stents, 1 uncoated stent migrated out and was not included in the dried mucous weight totals. This could explain the slightly higher mucous weight in the coated stents. Nevertheless, this current study demonstrates promising results in lowering airway injury in stents incorporated with the hydrophilic coating, and future studies, including a larger number of subjects, would be needed to corroborate our findings.

Creation and characterization of novel rat model for recessive dystrophic epidermolysis bullosa: Frameshift mutation of the Col7a1 gene leads to severe blistered phenotype.

Recessive dystrophic epidermolysis bullosa is a rare genodermatosis caused by a mutation of the Col7a1 gene. The Col7a1 gene codes for collagen type VII protein, a major component of anchoring fibrils. Mutations of the Col7a1 gene can cause aberrant collagen type VII formation, causing an associated lack or absence of anchoring fibrils. This presents clinically as chronic blistering, scarring, and fibrosis, often leading to the development of cutaneous squamous cell carcinoma. Patients also experience persistent pain and pruritus. Pain management and supportive bandaging remain the primary treatment options. The pathology of recessive dystrophic epidermolysis bullosa was first described in the 1980s, and there has since been a multitude of encouraging treatment options developed. However, in vivo research has been hindered by inadequate models of the disease. The various mouse models in existence possess longevity and surface area constraints, or do not adequately model a normal human disease state. In this paper, we describe a novel rat model of recessive dystrophic epidermolysis bullosa that offers an alternative to previous murine models. An 8-base pair deletion was induced in the Col7a1 gene of Lewis rats, which was subsequently found to cause a premature stop codon downstream. Homozygous mutants presented with a fragile and chronically blistered phenotype postnatally. Further histological analysis revealed subepidermal clefting and the absence of anchoring fibrils. The generation of this novel model offers researchers an easily maintained organism that possesses a larger surface area for experimental topical and transfused therapies to be tested, which may provide great utility in the future study of this debilitating disease.

Inhaled fosamprenavir for laryngopharyngeal reflux: Toxicology and fluid dynamics modeling.

Objectives: Approximately 25% of Americans suffer from laryngopharyngeal reflux (LPR), a disease for which no effective medical therapy exists. Pepsin is a predominant source of damage during LPR and a key therapeutic target. Fosamprenavir (FOS) inhibits pepsin and prevents damage in an LPR mouse model. Inhaled FOS protects at a lower dose than oral; however, the safety of inhaled FOS is unknown and there are no inhalers for laryngopharyngeal delivery. A pre-Good Lab Practice (GLP) study of inhaled FOS was performed to assess safety and computational fluid dynamics (CFD) modeling used to predict the optimal particle size for a laryngopharyngeal dry powder inhaler (DPI). Methods: Aerosolized FOS, amprenavir (APR), or air (control) were provided 5 days/week for 4 weeks (n = 6) in an LPR mouse model. Organs (nasal cavity, larynx, esophagus, trachea, lung, liver, heart, and kidney) were assessed by a pathologist and bronchoalveolar lavage cytokines and plasma cardiotoxicity markers were assessed by Luminex assay. CFD simulations were conducted in a model of a healthy 49-year-old female. Results: No significant increase was observed in histologic lesions, cytokines, or cardiotoxicity markers in FOS or APR groups relative to the control. CFD predicted that laryngopharyngeal deposition was maximized with aerodynamic diameters of 8.1-11.5 µm for inhalation rates of 30-60 L/min. Conclusions: A 4-week pre-GLP study supports the safety of inhaled FOS. A formal GLP assessment is underway to support a phase I clinical trial of an FOS DPI for LPR. Level of Evidence: NA.

Comparative dose effectiveness of intravenous and intrathecal AAV9.CB7.hIDS, RGX-121, in mucopolysaccharidosis type II mice.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disease caused by iduronate-2-sulfatase (IDS) deficiency, leading to accumulation of glycosaminoglycans (GAGs) and the emergence of progressive disease. Enzyme replacement therapy is the only currently approved treatment, but it leaves neurological disease unaddressed. Cerebrospinal fluid (CSF)-directed administration of AAV9.CB7.hIDS (RGX-121) is an alternative treatment strategy, but it is unknown if this approach will affect both neurologic and systemic manifestations. We compared the effectiveness of intrathecal (i.t.) and intravenous (i.v.) routes of administration (ROAs) at a range of vector doses in a mouse model of MPS II. While lower doses were completely ineffective, a total dose of 1 × 109 gc resulted in appreciable IDS activity levels in plasma but not tissues. Total doses of 1 × 1010 and 1 × 1011 gc by either ROA resulted in supraphysiological plasma IDS activity, substantial IDS activity levels and GAG reduction in nearly all tissues, and normalized zygomatic arch diameter. In the brain, a dose of 1 × 1011 gc i.t. achieved the highest IDS activity levels and the greatest reduction in GAG content, and it prevented neurocognitive deficiency. We conclude that a dose of 1 × 1010 gc normalized metabolic and skeletal outcomes, while neurologic improvement required a dose of 1 × 1011 gc, thereby suggesting the prospect of a similar direct benefit in humans.

Characterization of alkaline phosphatase cytochemistry in canine neoplastic and non-neoplastic pulmonary mass aspirates.

BACKGROUND: Distinguishing primary and secondary pulmonary neoplasms can be challenging via cytology, and a rapid, inexpensive diagnostic tool to differentiate these neoplasms is unavailable. Alkaline phosphatase cytochemistry (ALP-CC) has been used to identify primary pulmonary carcinomas in human patients, and we hypothesized it could be applied to canine lung aspirates. OBJECTIVE: We aimed to characterize ALP-CC expression in fine-needle aspirate (FNA) samples of canine pulmonary neoplastic and non-neoplastic tumors. METHODS: A retrospective case search was conducted to identify cases with contemporaneous cytology and histopathology reports from pulmonary lesions, including neoplastic and non-neoplastic etiologies. Slides prepared from pulmonary aspirates were stained for ALP-CC activity, and the percentage of ALP-CC-positive primary pulmonary epithelial tumors was determined. To characterize the ALP-CC expression in non-neoplastic cellular constituents of pulmonary FNA samples, mesothelial cells were also evaluated. RESULTS: Forty-eight canine cases met the inclusion criteria. ALP-CC-positive cells were seen in both neoplastic and non-neoplastic lesions. In non-neoplastic lesions, pulmonary epithelial cells were ALP-CC positive. Eighty-nine percent of primary pulmonary epithelial neoplasms were ALP-CC positive, and no ALP-CC positivity was noted in mesothelial cells. ALP-CC-positive neoplastic cells were seen in a metastatic amelanotic melanoma. CONCLUSIONS: Primary pulmonary epithelial neoplasms are frequently ALP-CC positive, but such positivity is not restricted to this tumor type. Non-neoplastic pulmonary epithelial cells can be ALP-CC positive, whereas mesothelial cells are negative.