Preclinical and clinical evaluation through serial colonoscopic evaluation of neratinib-induced diarrhea in HER2-positive breast cancer-A pilot study.

The irreversible pan-HER tyrosine kinase inhibitor neratinib is approved for patients with HER2-positive, early-stage and metastatic breast cancer (BC). Neratinib-associated diarrhea is the most common reason for early discontinuation. Preclinical studies identified mechanisms of neratinib-induced diarrhea and rationale for prophylactic and preventive measures. We studied effects of neratinib on rat intestines and conducted a phase 2 study of colon pathogenesis in patients with HER2-positive BC treated with neratinib (NCT04366713). Colon samples from female albino Wistar rats receiving neratinib or vehicle were examined for histopathological changes. Patients with HER2-positive BC received neratinib 240 mg once daily for up to 1 year. Colonoscopy biopsies were collected at baseline and at Day 28 to identify changes consistent with rat pathologies. Rat colons were markedly altered in appearance, with similar short circuit currents (Isc) and responses to carbachol and forskolin. Mucosal barrier loss and/or significant increase in secretory propensity in neratinib- versus control-treated animals were not seen. Two of four endpoint-evaluable patients presented with mild pathological changes, largely comparable with the rat model. Preclinical evidence supports an inflammatory component of neratinib-induced diarrhea without mucosal barrier function loss. Colonoscopy findings in patients with BC indicate mild or no pathological changes in the colon due to neratinib treatment.

Ferroptosis - a potential feature underlying neratinib-induced colonic epithelial injury.

PURPOSE: Neratinib, a small-molecule tyrosine kinase inhibitor (TKI) that irreversibly binds to human epidermal growth factor receptors 1, 2 and 4 (HER1/2/4), is an approved extended adjuvant therapy for patients with HER2-amplified or -overexpressed (HER2-positive) breast cancers. Patients receiving neratinib may experience mild-to-severe symptoms of gut toxicity including abdominal pain and diarrhoea. Despite being a highly prevalent complication in gut health, the biological processes underlying neratinib-induced gut injury, especially in the colon, remains unclear. METHODS: Real-time quantitative polymerase chain reaction (RT-qPCR) and histology were integrated to study the effect of, and type of cell death induced by neratinib on colonic tissues collected from female Albino Wistar rats dosed with neratinib (50 mg/kg) daily for 28 days. Additionally, previously published bulk RNA-sequencing and CRISPR-screening datasets on human glioblastoma SF268 cell line and glioblastoma T895 xenograft, and mouse TBCP1 breast cancer cell line were leveraged to elucidate potential mechanisms of neratinib-induced cell death. RESULTS: The severity of colonic epithelial injury, especially degeneration of surface lining colonocytes and infiltration of immune cells, was more pronounced in the distal colon than the proximal colon. Sequencing showed that apoptotic gene signature was enriched in neratinib-treated SF268 cells while ferroptotic gene signature was enriched in neratinib-treated TBCP1 cells and T895 xenograft. However, we found that ferroptosis, but less likely apoptosis, was a potential histopathological feature underlying colonic injury in rats treated with neratinib. CONCLUSION: Ferroptosis is a potential feature of neratinib-induced colonic injury and that targeting molecular machinery governing neratinib-induced ferroptosis may represent an attractive therapeutic approach to ameliorate symptoms of gut toxicity.

Targeting Acute Myeloid Leukemia Using Sphingosine Kinase 1 Inhibitor-Loaded Liposomes.

Acute myeloid leukemia (AML) kills 75% of patients and represents a major clinical challenge with a need to improve on current treatment approaches. Targeting sphingosine kinase 1 with a novel ATP-competitive-inhibitor, MP-A08, induces cell death in AML. However, limitations in MP-A08's "drug-like properties" (solubility, biodistribution, and potency) hinder its pathway to the clinic. This study demonstrates a liposome-based delivery system of MP-A08 that exhibits enhanced MP-A08 potency against AML cells. MP-A08-liposomes increased MP-A08 efficacy against patient AML cells (>140-fold) and significantly prolonged overall survival of mice with human AML disease (P = 0.03). The significant antileukemic property of MP-A08-liposomes could be attributed to its enhanced specificity, bioaccessibility, and delivery to the bone marrow, as demonstrated in the pharmacokinetic and biodistribution studies. Our findings indicate that MP-A08-liposomes have potential as a novel treatment for AML.

Haemostatic efficacy and inflammatory response of a novel beta-chitin patch in a cerebral small vessel injury model - A pilot study.

OBJECTIVE: Rapid and efficacious haemostasis is paramount in neurosurgery. Assessing the efficacy and short- and long-term safety of haemostatic agents utilised within cerebral tissue is essential. This pilot study investigates the haemostatic efficacy and long-term safety of a novel beta-chitin patch against traditionally used agents, bipolar and Floseal, within cerebral tissue. METHODS: Eighteen Merino sheep underwent standardised distal cortical vessel injury via temporal craniotomy. Sheep were randomised to receive 2 mls Floseal, 2 cm novel beta-chitin patch, or bipolar cautery to manage bleeding. All sheep underwent cerebral magnetic resonance imaging (MRI) at three months, before euthanasia and brain harvesting for histological assessment. RESULTS: Beta-chitin demonstrated a trend towards a faster mean time to haemostasis (TTH) compared to Floseal (223.3 ± 199 s v. 259.8 ± 186.4 s), albeit non-significant (p = 0.234). Radiologically, cerebrocortical necrosis (p = 0.842) and oedema (p = 0.368) were noted slightly more frequently in the beta-chitin group. Histologically, severe fibrotic (p = 0.017) and granulomatous changes at the craniotomy sites were only present in the beta-chitin group (p = 0.002). Neuronal degeneration was seen in all with Floseal, but beta-chitin showed a trend towards more severe reaction when present. Bipolar use predominantly showed an inflammatory cortical reaction with substantial microvascular proliferation, and Floseal showed worse severity and depth of subpial oedema, however no statistical significance was reached. CONCLUSION: All haemostats controlled bleeding, with beta-chitin demonstrating a non-inferior TTH compared to Floseal. However, it resulted in intense granulomatous and fibrotic changes, including degenerative neuronal reactions. More extensive studies are needed to assess these trends, to make further clinical inferences.

Alpha-B-Crystallin overexpression is sufficient to promote tumorigenesis and metastasis in mice.

BACKGROUND: αB-Crystallin is a heat shock chaperone protein which binds to misfolded proteins to prevent their aggregation. It is overexpressed in a wide-variety of cancers. Previous studies using human cancer cell lines and human xenograft models have suggested potential tumor promoter (oncogene) roles for αB-Crystallin in a wide-spectrum of cancers. METHODS: To determine the causal relationship between CRYAB overexpression and cancer, we generated a Cryab overexpression knock-in mouse model and monitor them for development of spontaneous and carcinogen (DMBA)-induced tumorigenesis. In order to investigate the mechanism of malignancies observed in this model multiple techniques were used such as immunohistochemical characterizations of tumors, bioinformatics analysis of publically available human tumor datasets, and generation of mouse embryonic fibroblasts (MEFs) for in vitro assays (clonogenic survival and migration assays and proteome analysis by mass-spectrometry). RESULTS: This model revealed that constitutive overexpression of Cryab results in the formation of a variety of lethal spontaneous primary and metastatic tumors in mice. In vivo, the overexpression of Cryab correlated with the upregulation of epithelial-to-mesenchymal (EMT) markers, angiogenesis and some oncogenic proteins including Basigin. In vitro, using E1A/Ras transformed MEFs, we observed that the overexpression of Cryab led to the promotion of cell survival via upregulation of Akt signaling and downregulation of pro-apoptotic pathway mediator JNK, with subsequent attenuation of apoptosis as assessed by cleaved caspase-3 and Annexin V staining. CONCLUSIONS: Overall, through the generation and characterization of Cryab overexpression model, we provide evidence supporting the role of αB-Crystallin as an oncogene, where its upregulation is sufficient to induce tumors, promote cell survival and inhibit apoptosis.

Efficacy and Safety of Novel Beta-Chitin Patches as Haemostat in Rat Vascular and Neurosurgical Model.

Background: Intraoperative hemorrhage is a major cause of poor post-operative outcome. Beta-chitin patch has previously been found to be an effective haemostat, but whether modifying the patch can improve its efficacy and safety, remains unknown. In this study, beta-chitin patches were modified using polyethylene oxide, Pluronic-F127 (Chi/F127), calcium (Chi/20%Ca), increased thickness (Chi/Thick) or polyphosphate (Chi/PP). Objective: Using rat (Wistar Albino; 8-10 weeks old) vascular and neurosurgical models, this project investigated and compared the efficacy and safety of beta-chitin patches with gauze, Surgicel and FloSeal. Methods: Ninety rats underwent a standardized femoral artery injury and were randomized to receive either beta-chitin patches, gauze, Surgicel or FloSeal. The bleeding time and total blood loss was measured. For the neurosurgical model, forty-four rats underwent a standardized cortical injury and randomization to a treatment group. Following a 48 h recovery period, their brains were collected for histopathological examination. Results: The mean bleeding time with Chitin (120.8 s) and Chi/PP (117.3 s) was ~60 s lower than Chi/F127, Chi/20%Ca and Chi/Thick (p < 0.05). Chitin and Chi/PP had a significantly lower bleeding time than FloSeal (174.2 s) (p < 0.05), but not Surgicel (172.7 s). Gauze (400 s) had a significantly higher bleeding time compared to all other groups (p < 0.05). There were no significant differences in the total blood loss between the groups. Histopathological examination of brains found no adverse inflammatory reaction to any of the haemostatic compounds. Conclusion: Chi/PP had superior haemostatic efficacy compared to Surgicel and FloSeal, but not compared to non-modified beta-chitin patch. All of the haemostats were equally safe.

Cep55 regulation of PI3K/Akt signaling is required for neocortical development and ciliogenesis.

Homozygous nonsense mutations in CEP55 are associated with several congenital malformations that lead to perinatal lethality suggesting that it plays a critical role in regulation of embryonic development. CEP55 has previously been studied as a crucial regulator of cytokinesis, predominantly in transformed cells, and its dysregulation is linked to carcinogenesis. However, its molecular functions during embryonic development in mammals require further investigation. We have generated a Cep55 knockout (Cep55-/-) mouse model which demonstrated preweaning lethality associated with a wide range of neural defects. Focusing our analysis on the neocortex, we show that Cep55-/- embryos exhibited depleted neural stem/progenitor cells in the ventricular zone as a result of significantly increased cellular apoptosis. Mechanistically, we demonstrated that Cep55-loss downregulates the pGsk3ß/ß-Catenin/Myc axis in an Akt-dependent manner. The elevated apoptosis of neural stem/progenitors was recapitulated using Cep55-deficient human cerebral organoids and we could rescue the phenotype by inhibiting active Gsk3ß. Additionally, we show that Cep55-loss leads to a significant reduction of ciliated cells, highlighting a novel role in regulating ciliogenesis. Collectively, our findings demonstrate a critical role of Cep55 during brain development and provide mechanistic insights that may have important implications for genetic syndromes associated with Cep55-loss.

The susceptibility of the aortic root: porcine aortic rupture testing under cardiopulmonary bypass.

BACKGROUND: In our earlier study on the functional limits of the aneurysmal aortic root we determined the pig root is susceptible to failure at high aortic pressures levels. We established a pig rupture model using cardiopulmonary bypass to determine the most susceptible region of the aortic root under the highest pressures achievable using continuous flow, and what changes occur in these regions on a macroscopic and histological level. This information may help guide clinical management of aortic root and ascending aorta pathology. METHODS: Five pigs underwent 4D flow MRI imaging pre surgery to determine vasopressor induced wall sheer stress and flow parameters. All pigs were then placed on cardiopulmonary bypass (CPB) via median sternotomy, and maximal aortic root and ascending aorta flows were initiated until rupture or failure, to determine the most susceptible region of the aorta. The heart was explanted and analysed histologically to determine if histological changes mirror the macroscopic observations. RESULTS: The magnetic resonance imaging (MRI) aortic flow and wall sheer stress (WSS) increased significantly in all regions of the aorta, and the median maximal pressures obtained during cardiopulmonary bypass was 497 mmHg and median maximal flows was 3.96 L/m. The area of failure in all experiments was the non-coronary cusp of the aortic valve. Collagen and elastin composition (%) was greatest in the proximal regions of the aorta. Collagen I and III showed greatest content in the inner aortic root and ascending aorta regions. CONCLUSIONS: This unique porcine model shows that the aortic root is most susceptible to failure at high continuous aortic pressures, supported histologically by different changes in collagen content and subtypes in the aortic root. With further analysis, this information could guide management of the aortic root in disease.

Histological regional analysis of the aortic root and thoracic ascending aorta: a complete analysis of aneurysms from root to arch.

BACKGROUND: Although aortic root and ascending aortic aneurysms are treated the same, they differ in embryological development and pathological processes. This study examines the microscopic structural differences between aortic root and ascending aortic aneurysms, correlating these features to the macroscopic pathophysiological processes. METHODS: We obtained surgical samples from ascending aortic aneurysms (n = 11), aortic root aneurysms (n = 3), and non-aneurysmal patients (n = 7), Aortic collagen and elastin content were examined via histological analysis, and immunohistochemistry techniques used to determine collagen I, III, and IV subtypes. Analysis was via observational features, and colour deconvolution quantification techniques. RESULTS: Elastin fiber disruption and fragmentation was the most extensive in the proximal aneurysmal regions. Medial fibrosis and collagen density increased in proximal aneurysmal regions and aortic root aneurysms (p < 0.005). Collagen I was seen in highest quantity in aortic root aneurysms. Collagen I content was greatest in the sinus tissue regions compared to the valvular and ostial regions (p < 0.005) Collagen III and IV quantification did not vary greatly. The most susceptible regions to ultrastructural changes in disease are the proximal ascending aorta and aortic root. CONCLUSIONS: The aortic root differs histologically from the ascending aorta confirming its unique composition in aneurysm pathology. These findings should prompt further evaluation on the influence of this altered structure on function which could potentially guide clinical management.

Prevention of adhesions post-abdominal surgery: Assessing the safety and efficacy of Chitogel with Deferiprone in a rat model.

INTRODUCTION: Adhesions are often considered to be an inevitable consequence of abdominal and pelvic surgery, jeopardizing the medium and long-term success of these procedures. Numerous strategies have been tested to reduce adhesion formation, however, to date, no surgical or medical therapeutic approaches have been successful in its prevention. This study demonstrates the safety and efficacy of Chitogel with Deferiprone and/or antibacterial Gallium Protoporphyrin in different concentrations in preventing adhesion formation after abdominal surgery. MATERIALS AND METHODS: 112 adult (8-10 week old) male Wistar albino rats were subjected to midline laparotomy and caecal abrasion, with 48 rats having an additional enterotomy and suturing. Kaolin (0.005g/ml) was applied to further accelerate adhesion formation. The abrasion model rats were randomized to receive saline, Chitogel, or Chitogel plus Deferiprone (5, 10 or 20 mM), together with Gallium Protoporphyrin (250µg/mL). The abrasion with enterotomy rats were randomised to receive saline, Chitogel or Chitogel with Deferiprone (1 or 5 mM). At day 21, rats were euthanised, and adhesions graded macroscopically and microscopically; the tensile strength of the repaired caecum was determined by an investigator blinded to the treatment groups. RESULTS: Chitogel with Deferiprone 5 mM significantly reduced adhesion formation (p<0.01) when pathologically assessed in a rat abrasion model. Chitogel with Deferiprone 5 mM and 1 mM also significantly reduced adhesions (p<0.05) after abrasion with enterotomy. Def-Chitogel 1mM treatment did not weaken the enterotomy site with treated sites having significantly better tensile strength compared to control saline treated enterotomy rats. CONCLUSIONS: Chitogel with Deferiprone 1 mM constitutes an effective preventative anti-adhesion barrier after abdominal surgery in a rat model. Moreover, this therapeutic combination of agents is safe and does not weaken the healing of the sutured enterotomy site.

Towards Human Translation of Lentiviral Airway Gene Delivery for Cystic Fibrosis: A One-Month CFTR and Reporter Gene Study in Marmosets.

Gene therapy continues to be a promising contender for the treatment of cystic fibrosis (CF) airway disease. We have previously demonstrated that airway conditioning with lysophosphatidylcholine (LPC) followed by delivery of a HIV-1-based lentiviral (LV) vector functionally corrects the CF transmembrane conductance regulator (CFTR) defect in the nasal airways of CF mice. In our earlier pilot study we showed that our technique can transduce marmoset lungs acutely; this study extends that work to examine gene expression in this nonhuman primate (NHP) 1 month after gene vector treatment. A mixture of three separate HIV-1 vesicular stomatitis virus G (VSV-G)-pseudotyped LV vectors containing the luciferase (Luc), LacZ, and hCFTR transgenes was delivered into the trachea through a miniature bronchoscope. We examined whether a single-dose delivery of LV vector after LPC conditioning could increase levels of transgene expression in the trachea and lungs compared with control (phosphate-buffered saline [PBS]) conditioning. At 1 month, bioluminescence was detected in vivo in the trachea of three of the six animals within the PBS control group, compared with five of the six LPC-treated animals. When examined ex vivo there was weak evidence that LPC improves tracheal Luc expression levels. In the lungs, bioluminescence was detected in vivo in four of the six PBS-treated animals, compared with five of the six LPC-treated animals; however, bioluminescence was present in all lungs when imaged ex vivo. LacZ expression was predominantly observed in the alveolar regions of the lung. hCFTR was detected by qPCR in the lungs of five animals. Basal cells were successfully isolated and expanded from marmoset tracheas, but no LacZ-positive colonies were detected. There was no evidence of an inflammatory response toward the LV vector at 1 month postdelivery, with cytokines remaining at baseline levels. In conclusion, we found weak evidence that LPC conditioning improved gene transduction in the trachea, but not in the marmoset lungs. We also highlight some of the challenges associated with translational lung gene therapy studies in NHPs.

The immunotoxicity, but not anti-tumor efficacy, of anti-CD40 and anti-CD137 immunotherapies is dependent on the gut microbiota.

Immune agonist antibodies (IAAs) are promising immunotherapies that target co-stimulatory receptors to induce potent anti-tumor immune responses, particularly when combined with checkpoint inhibitors. Unfortunately, their clinical translation is hampered by serious dose-limiting, immune-mediated toxicities, including high-grade and sometimes fatal liver damage, cytokine release syndrome (CRS), and colitis. We show that the immunotoxicity, induced by the IAAs anti-CD40 and anti-CD137, is dependent on the gut microbiota. Germ-free or antibiotic-treated mice have significantly reduced colitis, CRS, and liver damage following IAA treatment compared with conventional mice or germ-free mice recolonized via fecal microbiota transplant. MyD88 signaling is required for IAA-induced CRS and for anti-CD137-induced, but not anti-CD40-induced, liver damage. Importantly, antibiotic treatment does not impair IAA anti-tumor efficacy, alone or in combination with anti-PD1. Our results suggest that microbiota-targeted therapies could overcome the toxicity induced by IAAs without impairing their anti-tumor activity.

Proteomic analysis of nasal mucus samples of healthy patients and patients with chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) has a complex and multifactorial pathogenesis with a heterogeneous inflammatory profile. Proteomic analysis of nasal mucus may enable further understanding of protein abundances and biologic processes present in CRS and its endotypes compared with in healthy patients. OBJECTIVE: Our aim was to determine differences in the nasal mucus proteome of healthy patients and patients with CRS. METHODS: Nasal mucus was obtained from healthy patients, patients with CRS without nasal polyps (CRSsNP), and patients with CRS with nasal polyps (CRSwNP) before surgery. Gel electrophoresis was performed to fractionate the complex protein extracts before mass spectrometry analysis. Gene set enrichment analysis was performed on differentially expressed proteins. RESULTS: A total of 33 patients were included in this study (12 healthy, 10 with CRSsNP, and 11 with CRSwNP). In all, 1142 proteins were identified in mucus samples from healthy patients, 761 in mucus samples from patients with CRSsNP, and 998 in mucus samples from patients with CRSwNP. Dysfunction in immunologic pathways, reduced cellular signaling, and increased cellular metabolism with associated tissue remodeling pathways were present in patients with CRS compared with in healthy patients. CONCLUSION: Significant downregulation of mucosal immunity and antioxidant pathways with increased tissue modeling processes may account for the clinical manifestations of CRS. Ultimately, the differing proteome and biologic processes provide further insight into CRS pathogenesis and its endotypes.

Cep55 overexpression promotes genomic instability and tumorigenesis in mice.

High expression of centrosomal protein CEP55 has been correlated with clinico-pathological parameters across multiple human cancers. Despite significant in vitro studies and association of aberrantly overexpressed CEP55 with worse prognosis, its causal role in vivo tumorigenesis remains elusive. Here, using a ubiquitously overexpressing transgenic mouse model, we show that Cep55 overexpression causes spontaneous tumorigenesis and accelerates Trp53+/- induced tumours in vivo. At the cellular level, using mouse embryonic fibroblasts (MEFs), we demonstrate that Cep55 overexpression induces proliferation advantage by modulating multiple cellular signalling networks including the hyperactivation of the Pi3k/Akt pathway. Notably, Cep55 overexpressing MEFs have a compromised Chk1-dependent S-phase checkpoint, causing increased replication speed and DNA damage, resulting in a prolonged aberrant mitotic division. Importantly, this phenotype was rescued by pharmacological inhibition of Pi3k/Akt or expression of mutant Chk1 (S280A) protein, which is insensitive to regulation by active Akt, in Cep55 overexpressing MEFs. Moreover, we report that Cep55 overexpression causes stabilized microtubules. Collectively, our data demonstrates causative effects of deregulated Cep55 on genome stability and tumorigenesis which have potential implications for tumour initiation and therapy development.

Longitudinal Magnetic Resonance Spectroscopy and Diffusion Tensor Imaging in Sheep (Ovis aries) With Quinolinic Acid Lesions of the Striatum: Time-Dependent Recovery of N-Acetylaspartate and Fractional Anisotropy.

We created an excitotoxic striatal lesion model of Huntington disease (HD) in sheep, using the N-methyl-d-aspartate receptor agonist, quinolinic acid (QA). Sixteen sheep received a bolus infusion of QA (75 µL, 180 mM) or saline, first into the left and then (4 weeks later) into the right striatum. Magnetic resonance spectroscopy (MRS) and diffusion tensor imaging (DTI) of the striata were performed. Metabolite concentrations and fractional anisotropy (FA) were measured at baseline, acutely (1 week after each surgery) and chronically (5 weeks or greater after the surgeries). There was a significant decrease in the neuronal marker N-acetylaspartate (NAA) and in FA in acutely lesioned striata of the QA-lesioned sheep, followed by a recovery of NAA and FA in the chronically lesioned striata. NAA level changes indicate acute death and/or impairment of neurons immediately after surgery, with recovery of reversibly impaired neurons over time. The change in FA values of the QA-lesioned striata is consistent with acute structural disruption, followed by re-organization and glial cell infiltration with time. Our study demonstrates that MRS and DTI changes in QA-sheep are consistent with HD-like pathology shown in other model species and that the MR investigations can be performed in sheep using a clinically relevant human 3T MRI scanner.

A Novel Rat Model to Test Intra-Abdominal Anti-adhesive Therapy.

Background: Adhesion formation after abdominal surgery is considered almost inevitable and a major cause of morbidity. Novel treatments have been proposed, however there is a lack of suitable small animal models for pre-clinical evaluation, mainly due to inconsistency in adhesion formation in positive control animals. Here, we propose a new rat model of abdominal adhesions using Kaolin as the adhesion-inducing agent at an optimized dosage for testing newer agents in respect to their anti-adhesive property. Materials and Methods: Twenty-five adult (8-10 week old) male Wistar albino rats underwent midline laparotomy and caecal abrasion and were randomized to receive topical applications of normal saline or different concentrations and volumes of a Kaolin-based formulation. At day 14 rats were humanely killed, and adhesions graded macroscopically by an investigator blinded to the treatment groups, using pre-determined adhesion scores and microscopically using histopathology. Results: Kaolin at 0.005 g/mL caused consistent adhesions without compromising rat viability. At higher doses significant morbidity and mortality was observed in the animals treated. Conclusions: Kaolin induced adhesion in a rat abdominal surgery model is reliable and can be safely used to test the efficacy of novel anti-adhesive formulations to prevent intra-abdominal adhesions.

Phenotypic Characterization and Comparison of Cystic Fibrosis Rat Models Generated Using CRISPR/Cas9 Gene Editing.

Animal models of cystic fibrosis (CF) are essential for investigating disease mechanisms and trialing potential therapeutics. This study generated two CF rat models using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 gene editing. One rat model carries the common human Phe508del (ΔF508) CF transmembrane conductance regulator (CFTR) mutation, whereas the second is a CFTR knockout model. Phenotype was characterized using a range of functional and histologic assessments, including nasal potential difference to measure electrophysiological function in the upper airways, RNAscope in situ hybridization and quantitative PCR to assess CFTR mRNA expression in the lungs, immunohistochemistry to localize CFTR protein in the airways, and histopathologic assessments in a range of tissues. Both rat models revealed a range of CF manifestations, including reduced survival, intestinal obstruction, bioelectric defects in the nasal epithelium, histopathologic changes in the trachea, large intestine, and pancreas, and abnormalities in the development of the male reproductive tract. The CF rat models presented herein will prove useful for longitudinal assessments of pathophysiology and therapeutics.

Dietary sodium modulates nephropathy in Nedd4-2-deficient mice.

Salt homeostasis is maintained by tight control of Na+ filtration and reabsorption. In the distal part of the nephron the ubiquitin protein ligase Nedd4-2 regulates membrane abundance and thus activity of the epithelial Na+ channel (ENaC), which is rate-limiting for Na+ reabsorption. Nedd4-2 deficiency in mouse results in elevated ENaC and nephropathy, however the contribution of dietary salt to this has not been characterized. In this study we show that high dietary Na+ exacerbated kidney injury in Nedd4-2-deficient mice, significantly perturbing normal postnatal nephrogenesis and resulting in multifocal areas of renal dysplasia, increased markers of kidney injury and a decline in renal function. In control mice, high dietary Na+ resulted in reduced levels of ENaC. However, Nedd4-2-deficient kidneys maintained elevated ENaC even after high dietary Na+, suggesting that the inability to efficiently downregulate ENaC is responsible for the salt-sensitivity of disease. Importantly, low dietary Na+ significantly ameliorated nephropathy in Nedd4-2-deficient mice. Our results demonstrate that due to dysregulation of ENaC, kidney injury in Nedd4-2-deficient mice is sensitive to dietary Na+, which may have implications in the management of disease in patients with kidney disease.

Targeting neratinib-induced diarrhea with budesonide and colesevelam in a rat model.

PURPOSE: Neratinib is an irreversible pan-ErbB tyrosine kinase inhibitor used for the extended adjuvant treatment of early-stage HER2-positive breast cancer. Its use is associated with the development of severe diarrhea in up to 40% of patients in the absence of proactive management. We previously developed a rat model of neratinib-induced diarrhea and found inflammation and anatomical disruption in the ileum and colon. Here we tested whether anti-diarrheal interventions, budesonide and colesevelam, can reduce neratinib-induced diarrhea and intestinal pathology. METHODS: Rats were treated with 50 mg/kg neratinib via oral gavage for 14 or 28 days (total n = 64). Body weight and diarrhea severity were recorded daily. Apoptosis was measured using immunohistochemistry for caspase-3. Inflammation was measured via a multiplex cytokine/chemokine assay. ErbB levels were measured using PCR and Western Blot. RESULTS: Budesonide co-treatment caused rats to gain significantly less weight than neratinib alone from day 4 of treatment (P = 0.0418). Budesonide (P = 0.027) and colesevelam (P = 0.033) each reduced the amount of days with moderate diarrhea compared to neratinib alone. In the proximal colon, rats treated with neratinib had higher levels of apoptosis compared to controls (P = 0.0035). Budesonide reduced histopathological injury in the proximal (P = 0.0401) and distal colon (P = 0.027) and increased anti-inflammatory IL-4 tissue concentration (ileum; P = 0.0026, colon; P = 0.031) compared to rats treated with neratinib alone. In the distal ileum, while budesonide decreased ErbB1 mRNA expression compared to controls (P = 0.018) (PCR), an increase in total ErbB1 protein was detected (P = 0.0021) (Western Blot). CONCLUSION: Both budesonide and colesevelam show potential as effective interventions against neratinib-induced diarrhea.

Axonal spheroids in ovine neuroaxonal dystrophy are immunopositive to kinesin and dynein.

Neuroaxonal dystrophy (NAD) is a neurologic disorder of sheep characterized by accumulation of numerous axonal swellings (spheroids) in specific regions of the brainstem and spinal cord. Disruption of axonal transport, which is driven in anterograde and retrograde directions by the molecular motors, kinesin and dynein, respectively, is believed to contribute to spheroid development. Accordingly, we examined spheroids in ovine NAD cases immunohistochemically for kinesin and dynein and found both motor proteins, with dynein more strongly expressed than kinesin. Further investigations of the kinesin and dynein content of axonal spheroids in NAD, and other neurodegenerative disorders of domestic animals, could assist in better understanding the pathogenesis of these diseases.