The Interleukin-1 Receptor-Associated Kinase 4 Inhibitor PF-06650833 Blocks Inflammation in Preclinical Models of Rheumatic Disease and in Humans Enrolled in a Randomized Clinical Trial.

OBJECTIVE: To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. METHODS: Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. RESULTS: In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. CONCLUSION: These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.

41-Week Study of Progressive Diabetic Nephropathy in the ZSF1 fa/faCP Rat Model.

This symposium synopsis summarizes key results from a 41-week study of diabetic nephropathy (DN) in the type 2 diabetic ZSF1 fa/faCP rat model. During this study, we conducted longitudinal analysis of biomarkers, renal histopathology, ultrastructural assessment, renal quantitative image analysis, and transcriptome analysis of glomerular-enriched tissue. We concluded that there is translational value for using the ZSF1 rat model in mechanistic and therapeutic intervention efficacy studies for type 2 DN.

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.

Identification of Promising Urinary MicroRNA Biomarkers in Two Rat Models of Glomerular Injury.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate protein levels posttranscriptionally. miRNAs play important regulatory roles in many cellular processes and have been implicated in several diseases. Recent studies have reported significant levels of miRNAs in a variety of body fluids, raising the possibility that miRNAs could serve as useful biomarkers. Here, changes in miRNA expression patterns are described in 2 different rodent models of glomerular injury (acute puromycin aminonucleoside nephropathy and passive Heymann nephritis). By employing 2 different modes of glomerular insult, oxidative stress and immune-mediated toxicity, miRNA changes in both isolated glomeruli as well as urine specimens allow for identification of urinary miRNA biomarkers that are suggestive of drug-induced injury specifically to the glomerulus. Subsets of glomerular urinary miRNAs associated with these different modes of glomerular toxicity seem to be dependent on the mechanism of the induced injury, while 9 miRNAs that changed early in both glomerular and urine specimens were common to both studies. We further show that the miRNAs identified as mechanism-specific early glomerular injury biomarkers target key pathways and transcripts relevant to the type of insult, while the insult-independent changes might serve as ideal glomerular injury biomarkers.

Oral administration of soluble guanylate cyclase agonists to rats results in osteoclastic bone resorption and remodeling with new bone formation in the appendicular and axial skeleton.

Orally administered small molecule agonists of soluble guanylate cyclase (sGC) induced increased numbers of osteoclasts, multifocal bone resorption, increased porosity, and new bone formation in the appendicular and axial skeleton of Sprague-Dawley rats. Similar histopathological bone changes were observed in both young (7- to 9-week-old) and aged (42- to 46-week-old) rats when dosed by oral gavage with 3 different heme-dependent sGC agonist (sGCa) compounds or 1 structurally distinct heme-independent sGCa compound. In a 7-day time course study in 7- to 9-week-old rats, bone changes were observed as early as 2 to 3 days following once daily compound administration. Bone changes were mostly reversed following a 14-day recovery period, with complete reversal after 35 days. The mechanism responsible for the bone changes was investigated in the thyroparathyroidectomized rat model that creates a low state of bone modeling and remodeling due to deprivation of thyroid hormone, calcitonin (CT), and parathyroid hormone (PTH). The sGCa compounds tested increased both bone resorption and formation, thereby increasing bone remodeling independent of calciotropic hormones PTH and CT. Based on these studies, we conclude that the bone changes in rats were likely caused by increased sGC activity.

Identification of tubular injury microRNA biomarkers in urine: comparison of next-generation sequencing and qPCR-based profiling platforms.

BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNAs that regulate protein levels post-transcriptionally. miRNAs play important regulatory roles in many cellular processes and have been implicated in several diseases. Recent studies have reported significant levels of miRNAs in a variety of body fluids, raising the possibility that miRNAs could serve as useful biomarkers. Next-generation sequencing (NGS) is increasingly employed in biomedical investigations. Although concordance between this platform and qRT-PCR based assays has been reported in high quality specimens, information is lacking on comparisons in biofluids especially urine. Here we describe the changes in miRNA expression patterns in a rodent model of renal tubular injury (gentamicin). Our aim is to compare RNA sequencing and qPCR based miRNA profiling in urine specimen from control and rats with confirmed tubular injury. RESULTS: Our preliminary examination of the concordance between miRNA-seq and qRT-PCR in urine specimen suggests minimal agreement between platforms probably due to the differences in sensitivity. Our results suggest that although miRNA-seq has superior specificity, it may not detect low abundant miRNAs in urine samples. Specifically, miRNA-seq did not detect some sequences which were identified by qRT-PCR. On the other hand, the qRT-PCR analysis was not able to detect the miRNA isoforms, which made up the majority of miRNA changes detected by NGS. CONCLUSIONS: To our knowledge, this is the first time that miRNA profiling platforms including NGS have been compared in urine specimen. miRNAs identified by both platforms, let-7d, miR-203, and miR-320, may potentially serve as promising novel urinary biomarkers for drug induced renal tubular epithelial injury.

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.

Discovery of (3S,3aR)-2-(3-chloro-4-cyanophenyl)-3-cyclopentyl-3,3a,4,5-tetrahydro-2H-benzo[g]indazole-7-carboxylic acid (PF-3882845), an orally efficacious mineralocorticoid receptor (MR) antagonist for hypertension and nephropathy.

We have discovered a novel class of nonsteroidal pyrazoline antagonists of the mineralocorticoid receptor (MR) that show excellent potency and selectivity against other nuclear receptors. Early analogues were poorly soluble and had a propensity to inhibit the hERG channel. Remarkably, both of these challenges were overcome by incorporation of a single carboxylate moiety. Structural modification of carboxylate-containing lead R-4g with a wide range of substituents at each position of the pyrazoline ring resulted in R-12o, which shows excellent activity against MR and reasonable pharmacokinetic profile. Introduction of conformational restriction led to a novel series characterized by exquisite potency and favorable steroid receptor selectivity and pharmacokinetic profile. Oral dosing of 3S,3aR-27d (PF-3882845) in the Dahl salt sensitive preclinical model of salt-induced hypertension and nephropathy showed blood pressure attenuation significantly greater than that with eplerenone, reduction in urinary albumin, and renal protection. As a result of these findings, 3S,3aR-27d was advanced to clinical studies.

Neurological disease in wild loggerhead sea turtles Caretta caretta.

Beginning in October 2000, subadult loggerhead sea turtles Caretta caretta showing clinical signs of a neurological disorder were found in waters off south Florida, USA. Histopathology indicated generalized and neurologic spirorchiidiasis. In loggerhead sea turtles (LST) with neurospirorchiidiasis, adult trematodes were found in the meninges of the brain and spinal cord of 7 and 3 affected turtles respectively, and multiple encephalic intravascular or perivascular eggs were associated with granulomatous or mixed leukocytic inflammation, vasculitis, edema, axonal degeneration and occasional necrosis. Adult spirorchiids were dissected from meningeal vessels of 2 of 11 LST brains and 1 of 10 spinal cords and were identified as Neospirorchis sp. Affected LST were evaluated for brevetoxins, ciguatoxins, saxitoxins, domoic acid and palytoxin. While tissues from 7 of 20 LST tested positive for brevetoxins, the levels were not considered to be in a range causing acute toxicosis. No known natural (algal blooms) or anthropogenic (pollutant spills) stressors co-occurred with the turtle mortality. While heavy metal toxicosis and organophosphate toxicosis were also investigated as possible causes, there was no evidence for their involvement. We speculate that the clinical signs and pathologic changes seen in the affected LST resulted from combined heavy spirorchiid parasitism and possible chronic exposure to a novel toxin present in the diet of LST.

Potential sites of virus latency associated with indigenous pseudorabies viruses in feral swine.

Free-ranging feral swine (Sus scrofa) are known to be present in at least 32 states of the USA and are continuously expanding their range. Infection with pseudorabies virus (PRV) occurs in feral swine and the primary route of transmission in free-living conditions seems to be venereal. Between 1995 and 1999, naturally infected feral swine and experimentally infected hybrid progeny of feral and domestic swine, were kept in isolation and evaluated for occurrence of latent PRV indigenous to feral swine in sacral and trigeminal ganglia and tonsil. Sacral ganglia were shown, by polymerase chain reaction (PCR) amplification of the thymidine kinase (TK) gene of PRV, to be the most frequent sites of latency of PRV. Nine (56%) of 16 sacral ganglia, seven (44%) of 16 trigeminal ganglia, and five (39%) of 13 tonsils from naturally infected feral swine were positive for PCR amplification of TK sequences of PRV. These tissues were negative for PRV when viral isolation was attempted in Vero cells. DNA sequencing of cloned TK fragments from the sacral ganglia of two feral swine, showed only one nucleotide difference between the two fragments and extensive sequence homology to fragment sequences from various domestic swine PRV strains from China, Northern Ireland, and the USA. The hybrid feral domestic swine, experimentally inoculated with an indigenous feral swine PRV isolate by either the genital or respiratory route, acquired the infection but showed no clinical signs of pseudorabies. Virus inoculated into either the genital or respiratory tract could, at times, be isolated from both these sites. The most common latency sites were the sacral ganglia, regardless of the route and dose of infection in these experimentally infected hybrids. Nine of 10 sacral ganglia, six of 10 trigeminal ganglia, and three of 10 tonsils were positive for PCR amplification of TK sequences. No virus was isolated from these tissues in Vero cells. The demonstration of the sacral ganglia as the most common sites of latency of pseudorabies viruses indigenous to feral swine, supports the hypothesis that these viruses are primarily transmitted venereally, and not by the respiratory route as is common in domestic swine, in which the trigeminal ganglia are the predominant sites of virus latency.

An outbreak of fungal dermatitis and stomatitis in a free-ranging population of pigmy rattlesnakes (Sistrurus miliarius barbouri) in Florida.

Between September 1997 and March 1998, a severe skin, eye, and mouth disease was observed in a population of dusky pigmy rattlesnakes (Sistrurus miliarius barbouri), at the Lake Woodruff National Wildlife Refuge in Volusia County, Florida (USA). Three affected pigmy rattlesnakes were submitted for necropsy. All snakes had severe necrotizing and predominantly granulomatous dermatitis, stomatitis, and ophthalmitis, with involvement of the subadjacent musculature and other soft tissues. Numerous fungal hyphae were seen throughout tissue sections stained with periodic acid Schiff and Gomori's methenamine silver. Samples of lesions were cultured for bacteria and fungi. Based on hyphae and spore characteristics, four species of fungi were identified from culture: Sporothrix schenckii, Pestalotia pezizoides, Geotrichum candidum (Galactomyces geotrichum), and Paecilomyces sp. While no additional severely affected pigmy rattlesnakes were seen at the study site, a garter snake (Thamnophis sirtalis) and a ribbon snake (Thamnophis sauritis) with similar lesions were found. In 1998 and 1999, 42 pigmy rattlesnakes with multifocal minimal to moderate subcutaneous masses were seen at the study site. Masses from six of these snakes were biopsied in the field. Hyphae morphologically similar to those seen in the severe cases were observed with fungal stains. Analysis of a database representing 10,727 captures in previous years was performed after the 1998 outbreak was recognized. From this analysis we determined that 59 snakes with clinical signs similar to those seen during the 1998 outbreak were documented between 1992 and 1997. This study represents the first documented report of a mycotic disease of free-ranging snakes.

Pathogenicity of Haemoproteus danilewskyi, Kruse, 1890, in blue jays (Cyanocitta cristata).

Although the impact of blood parasite infections on passerine birds is potentially great, little is known of their pathologic effects. We studied Haemoproteus danilewskyi in experimentally infected captive and naturally infected free-ranging blue jays (Cyanocitta cristata) to determine patterns of infection and examine the pathologic effects of the parasite on the host. Physiologic changes, such as elevated numbers of lymphocytes, heterophils, basophils, eosinophils, and monocytes and decreased packed cell volume in the peripheral blood were associated with the erythrocytic phase of experimental infections of captive juvenile jays. Sublethal pathologic changes associated with the pre-erythrocytic phase of infections were observed in the liver, lung, and spleen. Schizonts were observed in the pulmonary capillaries of a 1 yr old jay necropsied 31 days post-inoculation, but not in 20 juvenile jays necropsied 57 days post-inoculation. In free-ranging naturally infected jays plasma protein concentration increased with density of natural infections.

Causes of mortality of free-ranging Florida panthers.

The Florida panther (Puma concolor coryi) is one of the most endangered mammals, with the entire population estimated to consist of only 30-50 adult animals. Between 1978 and 1999, 73 free-ranging Florida panther carcasses were submitted for postmortem evaluation, of which 47 (64%) were radiocollared and 26 (36%) were uncollared cats. Overall, mortality of panthers > 6-mo-old was due to vehicular trauma in 25 (35%), intraspecific aggression in 19 (26%), illegal kill in seven (10%), research activities in two (3%), infectious diseases in two (3%), esophageal tear in one (1%), pleuritis in one (1%), pyothorax in one (1%), aortic aneurysm in one (1%), atrial septal defect in one (1%), and causes of death were undetermined in 13 (18%) due to autolysis. Of the 25 panthers that were killed by vehicular trauma, 20 (80%) died between October and April. This coincides with increased number of winter visitors to south Florida. Among radiocollared panthers, intraspecific aggression was the primary cause of mortality for 19 (41%) dead cats. Of these cats, 16 (84%) were males and 14 (88%) were either less than 3 or more than 8-yr-old. These animals were probably fighting to establish or retain territory. Among the 26 uncollared panthers, vehicular trauma was the primary cause of mortality and was responsible for 16 (62%) deaths. This study documents the causes of mortality and the age, sex, and seasonal mortality trends for both radiocollared and uncollared free-ranging endangered Florida panthers over a 21-yr-period.

Retrospective study of proliferative papillary vulvitis in Florida panthers.

Proliferative, papillary vulvitis was identified in 16 of 34 (47%) free-ranging and captive female Florida panthers (Puma concolor coryi) monitored over a period from 1983-98. Gross lesions were characterized by extensive papilliferous proliferation in the mucosa of the vestibulum vaginae. Within lesions, the mean length and width of vestibular papillae were 1.07 +/- 0.39 mm (CV = 36%) and 0.55 +/- 0.11 mm (CV = 20%) respectively. Histologically, three to 12 layers of non-cornified stratified squamous epithelium with various degrees of basal cell spongiosis and rete ridge formation covered fibrous papillae. Mixed leukocytic mucosal inflammation also was observed. Infectious organisms were not observed, and immunohistochemical testing for the presence of papillomavirus antigens in specimens from seven panthers was negative. Lesions in nearly all of the panthers were first observed during a six-year period (1986-92), with one each in 1983, 1996 and 1998. There were no significant differences between the number of females having litters, the number of litters between age-matched and interval-matched females, and the interval between litters among lesions positive and lesion negative females over the 15 yr period. The severity of lesions did not appear to differ between parous and nulliparous free-ranging lesion-positive females. The cause of proliferative vulvitis remains unknown. However, the lesion did not appear to have a significant effect on reproduction.

Aplastic anemia associated with prolonged high-dose trimethoprim-sulfadiazine administration in two dogs.

Trimethoprim-sulfadiazine (TMP-SDZ) (Tribressin tablets 120 - 100 mg sulfadiazine, 20-mg trimethoprim [Coopers Animal Health, Inc., A Pitman-Moore Company, Mundelein, Ill.]) is a broad spectrum antibiotic combination effective in the treatment of bacterial pneumonia, urinary tract infections, pyoderma, meningitis, and prostatitis.(1) In clinical trials in puppies and adult dogs, TMP-SDZ was considered safe at both the manufacturer's recommended dose (15 mg/kg, b.i.d., or 30 mg/kg, u.i.d., per os for < 14 days(2)) and at 10 times that dose for 20 dayS.(3) Many infections, however, require prolonged high-dose therapy for resolution. The following study describes two cases of aplastic anemia and sepsis associated with intermittent, chronic (17-25 days), high-dose (25-30 mg/kg, b.i.d., per os) TMP-SDZ therapy recommended for the treatment of pyoderma.(4-7)