A modified mouse model of Friedreich's ataxia with conditional Fxn allele homozygosity delays onset of cardiomyopathy.

Friedreich's ataxia (FA) is an autosomal recessive disorder caused by a deficiency in frataxin (FXN), a mitochondrial protein that plays a critical role in the synthesis of iron-sulfur clusters (Fe-S), vital inorganic cofactors necessary for numerous cellular processes. FA is characterized by progressive ataxia and hypertrophic cardiomyopathy, with cardiac dysfunction as the most common cause of mortality in patients. Commonly used cardiac-specific mouse models of FA use the muscle creatine kinase (MCK) promoter to express Cre recombinase in cardiomyocytes and striated muscle cells in mice with one conditional Fxn allele and one floxed-out/null allele. These mice quickly develop cardiomyopathy that becomes fatal by 9-11 wk of age. Here, we generated a cardiac-specific model with floxed Fxn allele homozygosity (MCK-Fxnflox/flox). MCK-Fxnflox/flox mice were phenotypically normal at 9 wk of age, despite no detectable FXN protein expression. Between 13 and 15 wk of age, these mice began to display progressive cardiomyopathy, including decreased ejection fraction and fractional shortening and increased left ventricular mass. MCK-Fxnflox/flox mice began to lose weight around 16 wk of age, characteristically associated with heart failure in other cardiac-specific FA models. By 18 wk of age, MCK-Fxnflox/flox mice displayed elevated markers of Fe-S deficiency, cardiac stress and injury, and cardiac fibrosis. This modified model reproduced important pathophysiological and biochemical features of FA over a longer timescale than previous cardiac-specific mouse models, offering a larger window for studying potential therapeutics.NEW & NOTEWORTHY Previous cardiac-specific frataxin knockout models exhibit rapid and fatal cardiomyopathy by 9 wk of age. This severe phenotype poses challenges for the design and execution of intervention studies. We introduce an alternative cardiac-specific model, MCK-Fxnflox/flox, with increased longevity and delayed onset of all major phenotypes. These phenotypes develop to the same severity as previous models. Thus, this new model provides the same cardiomyopathy-associated mortality with a larger window for potential studies.

Zucker Lean Rats With Hepatic Steatosis Recapitulate Asymptomatic Metabolic Syndrome and Exhibit Greater Sensitivity to Drug-Induced Liver Injury Compared With Standard Nonclinical Sprague-Dawley Rat Model.

Fatty liver disease is a potential risk factor for drug-induced liver injury (DILI). Despite advances in nonclinical in vitro and in vivo models to assess liver injury during drug development, the pharmaceutical industry is still plagued by idiosyncratic DILI. Here, we tested the hypothesis that certain features of asymptomatic metabolic syndrome (namely hepatic steatosis) increase the risk for DILI in certain phenotypes of the human population. Comparison of the Zucker Lean (ZL) and Zucker Fatty rats fed a high fat diet (HFD) revealed that HFD-fed ZL rats developed mild hepatic steatosis with compensatory hyperinsulinemia without increases in liver enzymes. We then challenged steatotic HFD-fed ZL rats and Sprague-Dawley (SD) rats fed normal chow, a nonclinical model widely used in the pharmaceutical industry, with acetaminophen overdose to induce liver injury. Observations in HFD-fed ZL rats included increased liver injury enzymes and greater incidence and severity of hepatic necrosis compared with similarly treated SD rats. The HFD-fed ZL rats also had disproportionately higher hepatic drug accumulation, which was linked with abnormal hepatocellular efflux transporter distribution. Here, we identify ZL rats with HFD-induced hepatic steatosis as a more sensitive nonclinical in vivo test system for modeling DILI compared with SD rats fed normal chow.

Selective inhibition of BTK prevents murine lupus and antibody-mediated glomerulonephritis.

Autoantibody production and immune complex deposition within the kidney promote renal disease in patients with lupus nephritis. Thus, therapeutics that inhibit these pathways may be efficacious in the treatment of systemic lupus erythematosus. Bruton's tyrosine kinase (BTK) is a critical signaling component of both BCR and FcR signaling. We sought to assess the efficacy of inhibiting BTK in the development of lupus-like disease, and in this article describe (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxy]phenyl)-1H-pyrazole-4-carboxamide (PF-06250112), a novel highly selective and potent BTK inhibitor. We demonstrate in vitro that PF-06250112 inhibits both BCR-mediated signaling and proliferation, as well as FcR-mediated activation. To assess the therapeutic impact of BTK inhibition, we treated aged NZBxW_F1 mice with PF-06250112 and demonstrate that PF-06250112 significantly limits the spontaneous accumulation of splenic germinal center B cells and plasma cells. Correspondingly, anti-dsDNA and autoantibody levels were reduced in a dose-dependent manner. Moreover, administration of PF-06250112 prevented the development of proteinuria and improved glomerular pathology scores in all treatment groups. Strikingly, this therapeutic effect could occur with only a modest reduction observed in anti-dsDNA titers, implying a critical role for BTK signaling in disease pathogenesis beyond inhibition of autoantibody production. We subsequently demonstrate that PF-06250112 prevents proteinuria in an FcR-dependent, Ab-mediated model of glomerulonephritis. Importantly, these results highlight that BTK inhibition potently limits the development of glomerulonephritis by impacting both cell- and effector molecule-mediated pathways. These data provide support for evaluating the efficacy of BTK inhibition in systemic lupus erythematosus patients.

Mechanistic investigations of test article-induced pancreatic toxicity at the endocrine-exocrine interface in the rat.

Pancreatic toxicity commonly affects the endocrine or exocrine pancreas. However, it can also occur at the endocrine-exocrine interface (EEI), where the capillary network of the islet merges with the capillaries of the surrounding acinar tissue, that is, the insulo-acinar portal system. The goal of this article is to describe a novel, test article-induced pancreatic toxicity that originated at the EEI and to summarize investigations into the mechanistic basis of the injury. This injury was initially characterized by light microscopy in 7/14 day-toxicity studies in Sprague-Dawley (Crl: CD®[SD]) rats with undisclosed test articles. Microvascular injury at the interface resulted in peri-islet serum exudation, fibrin deposition, hemorrhage, inflammation, and secondary degeneration/necrosis of surrounding exocrine tissue. More chronic injury presented as islet fibrosis and lobular atrophy. Direct cytotoxicity affecting the capillary endothelium at the EEI was confirmed ultrastructurally on day 4. Endothelial microparticle and blood flow studies further confirmed endothelial involvement. Similar lesions occurred less frequently in 2 other rat strains and not in the mouse, dog, or cynomolgus macaque. In summary, in vivo and investigative study data confirmed primary endothelial cytotoxicity in the pathogenesis of this lesion and suggested that the lesion may be rat/rat strain-specific and of uncertain relevance for human safety risk assessment.

JAK inhibition with tofacitinib suppresses arthritic joint structural damage through decreased RANKL production.

OBJECTIVE: The mechanistic link between Janus kinase (JAK) signaling and structural damage to arthritic joints in rheumatoid arthritis (RA) is poorly understood. This study was undertaken to investigate how selective inhibition of JAK with tofacitinib (CP-690,550) affects osteoclast-mediated bone resorption in a rat adjuvant-induced arthritis (AIA) model, as well as human T lymphocyte RANKL production and human osteoclast differentiation and function. METHODS: Hind paw edema, inflammatory cell infiltration, and osteoclast-mediated bone resorption in rat AIA were assessed using plethysmography, histopathologic analysis, and immunohistochemistry; plasma and hind paw tissue levels of cytokines and chemokines (including RANKL) were also assessed. In vitro RANKL production by activated human T lymphocytes was evaluated by immunoassay, while human osteoclast differentiation and function were assessed via quantitative tartrate-resistant acid phosphatase staining and degradation of human bone collagen, respectively. RESULTS: Edema, inflammation, and osteoclast-mediated bone resorption in rats with AIA were dramatically reduced after 7 days of treatment with the JAK inhibitor, which correlated with reduced numbers of CD68/ED-1+, CD3+, and RANKL+ cells in the paws; interleukin-6 (transcript and protein) levels were rapidly reduced in paw tissue within 4 hours of the first dose, whereas it took 4-7 days of therapy for RANKL levels to decrease. Tofacitinib did not impact human osteoclast differentiation or function, but did decrease human T lymphocyte RANKL production in a concentration-dependent manner. CONCLUSION: These results suggest that the JAK inhibitor tofacitinib suppresses osteoclast-mediated structural damage to arthritic joints, and this effect is secondary to decreased RANKL production.

A protein kinase C α and ß inhibitor blunts hyperphagia to halt renal function decline and reduces adiposity in a rat model of obesity-driven type 2 diabetes.

Type 2 diabetes (T2D) and its complications can have debilitating, sometimes fatal consequences for afflicted individuals. The disease can be difficult to control, and therapeutic strategies to prevent T2D-induced tissue and organ damage are needed. Here we describe the results of administering a potent and selective inhibitor of Protein Kinase C (PKC) family members PKCα and PKCß, Cmpd 1, in the ZSF1 obese rat model of hyperphagia-induced, obesity-driven T2D. Although our initial intent was to evaluate the effect of PKCα/ß inhibition on renal damage in this model setting, Cmpd 1 unexpectedly caused a marked reduction in the hyperphagic response of ZSF1 obese animals. This halted renal function decline but did so indirectly and indistinguishably from a pair feeding comparator group. However, above and beyond this food intake effect, Cmpd 1 lowered overall animal body weights, reduced liver vacuolation, and reduced inguinal adipose tissue (iWAT) mass, inflammation, and adipocyte size. Taken together, Cmpd 1 had strong effects on multiple disease parameters in this obesity-driven rodent model of T2D. Further evaluation for potential translation of PKCα/ß inhibition to T2D and obesity in humans is warranted.

Multimodal biomarker investigation on efficacy and mechanism of action for the mammalian target of rapamycin inhibitor, temsirolimus, in a preclinical mammary carcinoma OncoMouse model: a translational medicine study in support for early clinical development.

The mammalian target of rapamycin (mTOR) has proven to be a valid therapeutic target in a number of human cancers, and it is a candidate for clinical trials in human breast cancer. We report on a biomarker-based translational medicine approach to assess the efficacy and mechanism of action for the mTOR inhibitor temsirolimus (CCI-779) in a mammary carcinoma OncoMouse model [polyomavirus middle T antigen (PyMT)]. The mTOR signaling pathway biomarkers were assessed using a reverse-phase protein array. Pharmacokinetics studies were conducted in both the tumor and plasma compartments. Pharmacodynamic biomarkers for compound-target engagement of tumor phospho-S6 proteins were assayed by Western blot. Temsirolimus (intravenously once a week for 2 weeks) was administered in both early and advanced stages of tumors. Biomarkers for temsirolimus effects on tumor progression were assessed by three-dimensional ultrasound imaging in combination with immunohistochemistry to assess vascular density (Texas red-dextran and CD31 immunostaining) and macrophage burden (F4/80 antigen). Tumor growth was significantly arrested in temsirolimus (25 ± 14% from 8 to 10 weeks, p < 0.05, and 26 ± 17% from 11 to 13 weeks, p < 0.01), compared with 493 ± 160 and 376 ± 50% increases, respectively, in vehicle-treated groups. Temsirolimus reduced tumor vascular density, 36 to 48 and 58 to 60%, p < 0.05, by the Texas red-dextran method or CD31-positive vessel count, respectively. Temsirolimus reduced tumor macrophage burden by 46% at 13 weeks (p < 0.05). Temsirolimus inhibited (p < 0.05) the phosphoproteins S6 pS235/236 and S6 pS240/244 up to 81 and 87%, respectively. We conclude that the multimodal biomarkers of temsirolimus efficacy and mechanism of action (phosphoproteins) strongly suggest that it might translate to therapeutic efficacy in human tumors that bear congruency to features present in the mammary carcinoma of PyMT tumors.

Experiments in the EpiDerm 3D Skin In Vitro Model and Minipigs In Vivo Indicate Comparatively Lower In Vivo Skin Sensitivity of Topically Applied Aneugenic Compounds.

Risk management of in vitro aneugens for topically applied compounds is not clearly defined because there is no validated methodology to accurately measure compound concentration in proliferating stratum basale keratinocytes of the skin. Here, we experimentally tested several known aneugens in the EpiDerm reconstructed human skin in vitro micronucleus assay and compared the results to flow cytometric mechanistic biomarkers (phospho-H3; MPM2, DNA content). We then evaluated similar biomarkers (Ki-67, nuclear area) using immunohistochemistry in skin sections of minipigs following topical exposure the potent aneugens, colchicine, and hesperadin. Data from the EpiDerm model showed positive micronucleus responses for all aneugens tested following topical or direct media dosing with similar sensitivity when adjusted for applied dose. Quantitative benchmark dose-response analysis exhibited increases in the mitotic index biomarkers phospho-H3 and MPM2 for tubulin binders and polyploidy for aurora kinase inhibitors are at least as sensitive as the micronucleus endpoint. By comparison, the aneugens tested did not induce histopathological changes, increases in Ki-67 immunolabeling or nuclear area in skin sections from the in vivo minipig study at doses in significant excess of those eliciting a response in vitro. Results indicate the EpiDerm in vitro micronucleus assay is suitable for the hazard identification of aneugens. The lack of response in the minipig studies indicates that the barrier function of the minipig skin, which is comparable to human skin, protects from the effects of aneugens in vivo. These results provide a basis for conducting additional studies in the future to further refine this understanding.

Pharmacogenomic, physiological, and biochemical investigations on safety and efficacy biomarkers associated with the peroxisome proliferator-activated receptor-gamma activator rosiglitazone in rodents: a translational medicine investigation.

Peroxisome proliferator-activated receptor (PPAR)-gamma modulators, a class of antidiabetic drugs, have been associated with cardiovascular risks in type 2 diabetes in humans. The objective of this study was to explore possible cardiovascular risk biomarkers associated with PPAR-gamma in rodents that could provide an alert for risk to humans. Normal, myocardial infarction-induced heart failure (HF) or Zucker diabetic fatty (ZDF) rats were used. Rats (n = 5-6) were treated with either vehicle or rosiglitazone (RGZ; 3 or 45 mg/kg/day p.o.) for 4 weeks. Biomarkers for potential cardiovascular risks were assessed, including 1) ultrasound for cardiac structure and function; 2) neuroendocrine and hormonal plasma biomarkers of cardiovascular risk; 3) pharmacogenomic profiling of cardiac and renal tissue by targeted tissue low-density gene array representing ion channels and transporters, and components of the renin-angiotensin-aldosterone system; and 4) immunohistochemistry for cardiac fibrosis, hypertrophy, and inflammation (macrophages and tumor necrosis factor-alpha). HF was confirmed by increase in cardiac brain natriuretic peptide expression (p < 0.01) and echocardiography. Adequate exposure of RGZ was confirmed by pharmacokinetics (plasma drug levels) and the pharmacodynamic biomarker adiponectin. In normal or HF rats, RGZ had no negative effects on any of the biomarkers investigated. Similarly, RGZ had no significant effects on gene expression except for the increase in interleukin-6 mRNA expression in the heart and decrease in epithelial sodium channel beta in the kidney. In contrast, echocardiography showed improved cardiac structure and function after RGZ in ZDF rats. Taken together, this study suggests a limited predictive power of these preclinical models in respect to observed clinical adverse effects associated with RGZ.

Correlation between 2- and 3-dimensional assessment of tumor volume and vascular density by ultrasonography in a transgenic mouse model of mammary carcinoma.

OBJECTIVE: Visualization and quantification of angiogenesis are instrumental in development of antiangiogenic therapy. Although both 2-dimensional (2D) and 3-dimensional (3D) ultrasonography have been used to monitor tumor growth and vasculature development, the correlation between them has not been sufficiently investigated. We hereby investigated the 2D and 3D sonographic correlation for tumor volume and vascular density confirmed by histologic assessment in the polyoma virus middle T antigen (PyMT) mouse model of mammary carcinoma. METHODS: Female PyMT mouse tumors were evaluated by ultrasonography in the 2D region of interest (ROI), 3D tumor volume, and 2D and 3D microvascular density after a bolus infusion of a nontargeted contrast-enhanced microbubble agent. Texas Red-dextran was used for quantitative histologic assessment of the tumor microvascular density. RESULTS: The individual 2D tumor ROI area correlated with the 3D tumor volume throughout the 2-week period. However, the extent of the increase in the 3D volume (380%; P < .01; n = 10) was higher than that of the 2D ROI area (72%; P < .01; n = 8-11). A significant and comparable increase in vascular density accessed by both 2D (87%; P < .05; n = 8) and 3D (64%; P < .05; n = 8) imaging was documented. Vascular density obtained through 3D imaging correlated significantly with 2D measurement. These data were confirmed by Texas Red-dextran quantification of vascular density. Conclusions. This study showed a valid application of sonographically based imaging technology in tumor volume and vascular density assessment as well as their 2D and 3D correlation, of which tumor vascular density measured by 2D ultrasonography appeared to be better correlated with the 3D data. Our data indicate that ultrasonography can be applied for real-time, accurate, noninvasive imaging of the tumor volume and vascular density in preclinical models.

Thrombospondin-I is a critical modulator in non-alcoholic steatohepatitis (NASH).

Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease characterized by dysregulated lipid metabolism and chronic inflammation ultimately resulting in fibrosis. Untreated, NAFLD may progress to non-alcoholic steatohepatitis (NASH), cirrhosis and death. However, currently there are no FDA approved therapies that treat NAFLD/NASH. Thrombospondin-I (TSP-1) is a large glycoprotein in the extracellular matrix that regulates numerous cellular pathways including transforming growth factor beta 1 (TGF-ß1) activation, angiogenesis, inflammation and cellular adhesion. Increased expression of TSP-1 has been reported in various liver diseases; however, its role in NAFLD/NASH is not well understood. We first examined TSP-1 modulation in hepatic stellate cell activation, a critical initiating step in hepatic fibrosis. Knockdown or inhibition of TSP-1 attenuated HSC activation measured by alpha smooth muscle actin (α-SMA) and Collagen I expression. To investigate the impact of TSP-1 modulation in context of NAFLD/NASH, we examined the effect of TSP-1 deficiency in the choline deficient L-amino acid defined high fat diet (CDAHFD) model of NASH in mice by assessing total body and liver weight, serum liver enzyme levels, serum lipid levels, liver steatosis, liver fibrosis and liver gene expression in wild type (WT) and TSP-1 null mice. CDAHFD fed mice, regardless of genotype, developed phenotypes of NASH, including significant increase in liver weight and liver enzymes, steatosis and fibrosis. However, in comparison to WT, CDAHFD-fed TSP-1 deficient mice were protected against numerous NASH phenotypes. TSP-1 null mice exhibited a decrease in serum lipid levels, inflammation markers and hepatic fibrosis. RNA-seq based transcriptomic profiles from the liver of CDAHFD fed mice determined that both WT and TSP-1 null mice exhibited similar gene expression signatures following CDAHFD, similar to biophysical and histological assessment comparison. Comparison of transcriptomic profiles based on genotype suggested that peroxisome proliferator activated receptor alpha (PPARα) pathway and amino acid metabolism pathways are differentially expressed in TSP-1 null mice. Activation of PPARα pathway was supported by observed decrease in serum lipid levels. Our findings provide important insights into the role of TSP-1 in context of NAFLD/NASH and TSP-1 may be a target of interest to develop anti-fibrotic therapeutics for NAFLD/NASH.

Molecular Profiling of Ulcerative Colitis Subjects from the TURANDOT Trial Reveals Novel Pharmacodynamic/Efficacy Biomarkers.

BACKGROUND AND AIMS: To define pharmacodynamic and efficacy biomarkers in ulcerative colitis [UC] patients treated with PF-00547659, an anti-human mucosal addressin cell adhesion molecule-1 [MAdCAM-1] monoclonal antibody, in the TURANDOT study. METHODS: Transcriptome, proteome and immunohistochemistry data were generated in peripheral blood and intestinal biopsies from 357 subjects in the TURANDOT study. RESULTS: In peripheral blood, C-C motif chemokine receptor 9 [CCR9] gene expression demonstrated a dose-dependent increase relative to placebo, but in inflamed intestinal biopsies CCR9 gene expression decreased with increasing PF-00547659 dose. Statistical models incorporating the full RNA transcriptome in inflamed intestinal biopsies showed significant ability to assess response and remission status. Oncostatin M [OSM] gene expression in inflamed intestinal biopsies demonstrated significant associations with, and good accuracy for, efficacy, and this observation was confirmed in independent published studies in which UC patients were treated with infliximab or vedolizumab. Compared with the placebo group, intestinal T-regulatory cells demonstrated a significant increase in the intermediate 22.5-mg dose cohort, but not in the 225-mg cohort. CONCLUSIONS: CCR9 and OSM are implicated as novel pharmacodynamic and efficacy biomarkers. These findings occur amid coordinated transcriptional changes that enable the definition of surrogate efficacy biomarkers based on inflamed biopsy or blood transcriptomics data.ClinicalTrials.gov identifierNCT01620255.

Quantitative biodistribution of biotherapeutics at whole body, organ and cellular levels by autoradiography.

AIM: Tools for mapping and quantifying monoclonal antibody (mAb) and peptide biotherapeutics distribumtion were evaluated by comparing data from three independent methods conducted at the whole body, organ or tissue, and cellular levels. MATERIALS & METHODS: [3H]-mAb1 and [3H]-peptide A were administered intravenously to rats followed by quantitative whole-body autoradiography, kidney macro-autoradiography and micro-autoradiography. RESULTS: [3H]-mAb1 and [3H]-peptide A concentrations were measured in anatomical regions ranging from whole body to whole organ to sub-organ level, such as the kidney glomerulus, with increasing resolution. The tissue/blood [3H]-mAb1 concentrations in selected kidney microenvironments were comparable among the three quantitative methods. CONCLUSION: Quantitative whole-body autoradiography, tissue macro-autoradiography and micro-autoradiography all provide useful tools for quantifying the concentrations of biotherapeutics at different anatomical levels in tissues, facilitating better predictions of efficacy and toxicity.

CXCL10/CXCR3 axis is associated with disease activity and the development of mucocutaneous lesions in patients with Behçet's disease.

The objective of this study was to investigate CXC chemokines and its receptor in patients with Behcet's disease (BD) and their associations with disease activity. Blood samples were collected from 109 BD patients and 36 age- and sex-matched healthy controls (HCs). Twenty-two follow-up blood samples were collected in BD patients. Serum CXC chemokines (CXCL1, CXCL8, CXCL9, CXCL10, CXCL12, CXCL13 and CXCL16) and cell surface marker expression (CD3, CD4 and CXCR3) in peripheral blood mononuclear cells (PBMCs) were assayed. Clinical features including disease activity were evaluated at the time of blood collection. CXCR3 expression in skin and intestinal lesions from BD patients and HCs was assessed via immunohistochemistry. Serum CXCL10 levels were correlated with disease activity in terms of Behçet's Disease Current Activity Form (BDCAF) (p < 0.001). In follow-up BD patients, changes in serum CXCL10 levels tended to be correlated with those of BDCAF. The percentage of CXCR3 expression in CD3-positive cells in PBMCs was inversely correlated with serum CXCL10 levels in BD patients (p = 0.022). By immunohistochemistry, the number of CXCR3-positive mononuclear cells was higher in skin and intestinal lesions of BD patients than in those of HCs. These results suggest that the CXCL10/CXCR3 axis may contribute to the pathogenesis of BD.

High resolution molecular and histological analysis of renal disease progression in ZSF1 fa/faCP rats, a model of type 2 diabetic nephropathy.

ZSF1 rats exhibit spontaneous nephropathy secondary to obesity, hypertension, and diabetes, and have gained interest as a model system with potentially high translational value to progressive human disease. To thoroughly characterize this model, and to better understand how closely it recapitulates human disease, we performed a high resolution longitudinal analysis of renal disease progression in ZSF1 rats spanning from early disease to end stage renal disease. Analyses included metabolic endpoints, renal histology and ultrastructure, evaluation of a urinary biomarker of fibrosis, and transcriptome analysis of glomerular-enriched tissue over the course of disease. Our findings support the translational value of the ZSF1 rat model, and are provided here to assist researchers in the determination of the model's suitability for testing a particular mechanism of interest, the design of therapeutic intervention studies, and the identification of new targets and biomarkers for type 2 diabetic nephropathy.

Understanding Lung Deposition of Alpha-1 Antitrypsin in Acute Experimental Mouse Lung Injury Model Using Fluorescence Microscopy.

Human plasma-derived α1-antitrypsin (AAT) delivered by intravenous infusion is used as augmentation therapy in patients with emphysema who have a genetic mutation resulting in deficiency of AAT. Inhalation is an alternative route of administration that can potentially increase the efficacy and convenience of treatment. This study was conducted to determine whether delivery to the lungs, initially via the intratracheal (IT) route of administration, would deliver efficacious levels of a recombinant AAT (rAAT) to the site of action in the lungs in mice. 125I-radiolabeled rAAT, fluorophore-conjugated rAAT (rAAT-Alexa488), and NE680 (neutrophil elastase 680, a silent fluorescent substrate of neutrophil elastase which fluoresces in the near-infrared range upon activation by neutrophil elastase) were used to characterize the pharmacokinetics and tissue distribution profile, distribution of rAAT within the lung, and efficacy of rAAT to inhibit neutrophil elastase at the site of action, respectively. The study has demonstrated that rAAT was able to gain access to locations where neutrophil elastase was localized. The histochemical quantification of rAAT activity relative to dose at the site of action provided here will improve confidence in predicting the human dose via the inhalation route.

Soluble Fn14 Is Detected and Elevated in Mouse and Human Kidney Disease.

The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tissue injury to mediate local tissue responses including inflammation and tissue remodeling. We found that in various models of kidney disease, Fn14 expression (mRNA and protein) is upregulated in the kidney. These models include: lupus nephritis mouse models (Nephrotoxic serum Transfer Nephritis and MRL.Faslpr/lpr), acute kidney injury models (Ischemia reperfusion injury and Folic acid injury), and a ZSF-1 diabetic nephropathy rat model. Fn14 expression levels correlate with disease severity as measured by disease histology. We have also shown for the first time the detection of soluble Fn14 (sFn14) in the urine and serum of mice. Importantly, we found the sFn14 levels are markedly increased in the diseased mice and are correlated with disease biomarkers including proteinuria and MCP-1. We have also detected sFn14 in human plasma and urine. Moreover, sFn14 levels, in urine are significantly increased in DN patients and correlated with proteinuria and MCP-1 levels. Thus our data not only confirm the up-regulation of Fn14/TWEAK pathway in kidney diseases, but also suggest a novel mechanism for its regulation by the generation of sFn14. The correlation of sFn14 levels and disease severity suggest that sFn14 may serve as a potential biomarker for both acute and chronic kidney diseases.

Inhibition of factor Xa reduces ischemic brain damage after thromboembolic stroke in rats.

BACKGROUND AND PURPOSE: Factor Xa (FXa) is a key coagulation protease and target for novel antithrombotic agents for prevention and treatment of diverse thromboembolic disorders. In the present study we describe the effect of a novel, potent, and selective FXa inhibitor, DPC602, on brain damage and neurobehavioral consequence in a rat thromboembolic model of stroke. METHODS: Thromboembolic stroke was induced in rats by placement of an autologous clot into the middle cerebral artery. RESULTS: Laser-Doppler monitoring of cerebral blood flow demonstrated that DPC602 (8 mg/kg, single IV/IP bolus pretreatment) markedly improved cerebral blood flow after thromboembolic stroke by 25% to 160% (n=6; P<0.001) at 1 to 6 hours. DPC602 demonstrated concentration- and time-dependent reductions in infarct size, with maximal effect (89% reduction; n=14; P<0.001) at the highest dose over controls. Neurological function was also significantly improved in DPC602-treated rats at days 1, 3, and 7 (n=13; P<0.01). DPC602 treatment did not cause cerebral hemorrhage, assessed by free hemoglobin in the ischemic brain tissues. CONCLUSIONS: These data suggest that anticoagulation with a selective FXa inhibitor might ameliorate the extent of ischemic brain damage and neurological deficits after a thromboembolic event. Enhanced clot dissolution and early reperfusion may account for the cerebrovascular-protective effect of the drug.

Maternal deprivation increases cell death in the infant rat brain.

Prolonged separation from the mother can interfere with normal growth and development and is a significant risk factor for adult psychopathology. In rodents, separation of a pup from its mother increases the behavioral and endocrine responses to stress for the lifetime of the animal. Here we investigated whether maternal deprivation could affect brain development of infant rats via changes in the rate of cell death as measured by labeling the 3' end of DNA fragments using terminal transferase (ApopTag). At postnatal day 12 (P12), the number of cells undergoing cell death approximately doubled in the cerebral cortex, cerebellar cortex and in several white matter tracts following 24 h of maternal deprivation. Deprivation strongly increased the number of ApopTag-labeled cells at P6 but not at P20. Stroking the infant rats only partially reversed the effects of maternal deprivation. Increased cell death in white matter tracts correlated with an induction of nerve growth factor which has been previously associated with oligodendrocyte cell death. Cell birth was either unchanged or decreased in response to deprivation. These results indicate that maternal deprivation can alter normal brain development by increasing cell death of neurons and glia, and provides a potential mechanism by which early environmental stressors may influence subsequent behavior.

Recombinant adenoviral expression of dominant negative IkappaBalpha protects brain from cerebral ischemic injury.

Transcription factor NF-kappaB is associated with inflammatory response and cell survival. Under inactive condition, NF-kappaB is sequestered in the cytoplasm by an anchor protein, inhibitor of NF-kappaB (IkappaB). NF-kappaB was shown to be activated during ischemic brain injury. In the present study we have investigated the role of NF-kappaB in ischemic brain injury using a recombinant adenovirus expressing a dominant negative form of IkappaB (Adv/IkappaBdn) to specifically inhibit NF-kappaB activation. Our data demonstrated that cortical injection of Adv/IkappaBdn significantly reduced ischemic brain injury following permanent occlusion of the middle cerebral artery (MCAO) in rats, showing 55% reduction (p<0.01, n=8) in total ischemic lesion or 80% reduction (p<0.001) in the cortical area with Adv/IkappaBdn expression. Similarly, Adv/IkappaBdn expression significantly decreased neurological deficits (37% reduction over controls, p<0.01, n=8). These data provide further evidence for the role of NF-kappaB/IkappaB in ischemic brain injury and suggest that inhibition of NF-kappaB is neuroprotective in focal stroke.