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Pulmonary arterial hypertension (PAH) is a progressive disease defined by a chronic elevation in pulmonary arterial pressure with extensive pulmonary vascular remodeling and perivascular inflammation characterized by an accumulation of macrophages, lymphocytes, dendritic cells, and mast cells. Although the exact etiology of the disease is unknown, clinical as well as preclinical data strongly implicate a role for serotonin (5-HT) in the process. Here, we investigated the chronic effects of pharmacological inhibition of tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in peripheral 5-HT biosynthesis, in two preclinical models of pulmonary hypertension (PH), the monocrotaline (MCT) rat and the semaxanib (SUGEN, Medinoah, Suzhou, China)-hypoxia rat. In both PH models, ethyl (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1'-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate and ethyl (S)-8-(2-amino-6-((R)-1-(3',4'-dimethyl-3-(3-methyl-1 H-pyrazol-1-yl)-[1,1'-biphenyl]-4-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate, novel orally active TPH1 inhibitors with nanomolar in vitro potency, decreased serum, gut, and lung 5-HT levels in a dose-dependent manner and significantly reduced pulmonary arterial pressure, and pulmonary vessel wall thickness and occlusion in male rats. In the MCT rat model, decreases in lung 5-HT significantly correlated with reductions in histamine levels and mast cell number (P < 0.001, r 2 = 0.88). In contrast, neither ambrisentan nor tadalafil, which are vasodilators approved for the treatment of PAH, reduced mast cell number or 5-HT levels, nor were they as effective in treating the vascular remodeling as were the TPH1 inhibitors. When administered in combination with ambrisentan, the TPH1 inhibitors showed an additive effect on pulmonary vascular remodeling and pressures. These data demonstrate that in addition to reducing vascular remodeling, TPH1 inhibition has the added benefit of reducing the perivascular mast cell accumulation associated with PH., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

As a follow-up to the discovery of our spirocyclic proline-based TPH1 inhibitor lead, we describe the optimization of this scaffold. Through a combination of X-ray co-crystal structure guided design and an in vivo screen, new substitutions in the lipophilic region of the inhibitors were identified. This effort led to new TPH1 inhibitors with in vivo efficacy when dosed as their corresponding ethyl ester prodrugs. In particular, 15b (KAR5585), the prodrug of the potent TPH1 inhibitor 15a (KAR5417), showed robust reduction of intestinal serotonin (5-HT) levels in mice. Furthermore, oral administration of 15b generated high and sustained systemic exposure of the active parent 15a in rats and dogs. KAR5585 was selected for further pharmacological evaluation in disease models associated with a dysfunctional peripheral 5-HT system., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

An increasing number of diseases have been linked to a dysfunctional peripheral serotonin system. Given that tryptophan hydroxylase 1 (TPH1) is the rate limiting enzyme in the biosynthesis off serotonin, it represents an attractive target to regulate peripheral serotonin. Following up to our first disclosure, we report a new chemotype of TPH1 inhibitors where-by the more common central planar heterocycle has been replaced with an open-chain, acyl guanidine surrogate. Through our work, we found that compounds of this nature provide highly potent TPH1 inhibitors with favorable physicochemical properties that were effective in reducing murine intestinal 5-HT in vivo. Furthermore, we obtained a high resolution (1.90Å) X-ray structure crystal structure of one of these inhibitors (compound 51) that elucidated the active conformation along with revealing a dimeric form of TPH1 for the first time., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aim: To determine if a noninvasive complementary therapy, Healing Touch, would benefit women undergoing diagnostic procedures for the determination of breast cancer. Women often experience high levels of fear and anxiety during this diagnostic period., Study Design: A randomized controlled pilot study., Method: An out-patient clinic specializing in breast care management was used. Seventy-three women age 18 to 85 years old participated, with 31 in the control group of standard care and 42 in the intervention group receiving Healing Touch, a noninvasive energy therapy. A specific technique, magnetic clearing, was provided by a practitioner for 15 minutes prior to the biopsy procedure. Both the State-Trait Anxiety Inventory and the Coping Resources Inventory were used preprocedurally and the following day to assess changes., Results: A mixed analysis of variance indicated that State Anxiety for the Healing Touch group showed a statistically significant reduction of anxiety that was sustained into the following day, F(2, 142) = 10.94, p < .001. For Trait Anxiety, there was a significant change pre-and postintervention to the day after, F(2, 142) = 5.15, p < .007. The Coping Resources Inventory had significant changes in two subcategories, Emotional, F(2, 142) = 6.10, p = .003, and the Spiritual/Philosophical, F(2, 142) = 6.10, p < .001, in the Healing Touch group., Conclusion: Healing Touch may have benefit in reducing anxiety from diagnostic breast procedures., (© The Author(s) 2015.)

The central role of the biogenic monoamine serotonin (5-hydroxytryptamine, 5-HT) as a neurotransmitter with important cognitive and behavioral functions is well known. However, 5-HT produced in the brain only accounts for approximately 5% of the total amount of 5-HT generated in the body. At the onset of our work, it appeared that substituted phenylalanine derivatives or related aryl amino acids were required to produce potent inhibitors of TPH1, as significant losses of inhibitory activity were noted in the absence of this structural element. We disclose herein the discovery of a new class of TPH1 inhibitors that significantly lower peripherally 5-HT., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Wild water birds are the natural reservoir for low-pathogenic avian influenza viruses (AIV). However, our ability to investigate the epizootiology of AIV in these migratory populations is challenging and, despite intensive worldwide surveillance, remains poorly understood. We conducted a cross-sectional, retrospective analysis in Pacific Flyway Lesser Snow Geese, Chen caerulescens, to investigate AIV serology and infection patterns. We collected nearly 3000 sera samples from Snow Geese at two breeding colonies in Russia and Canada during 1993-1996 and swab samples from >4000 birds at wintering and migration areas in the United States during 2006-2011. We found seroprevalence and annual seroconversion varied considerably among years. Seroconversion and infection rates also differed between Snow Goose breeding colonies and wintering areas, suggesting that AIV exposure in this gregarious waterfowl species is likely occurring during several phases (migration, wintering, and potentially breeding areas) of the annual cycle. We estimated AIV antibody persistence was longer (14 months) in female geese compared to males (6 months). This relatively long period of AIV antibody persistence suggests that subtype-specific serology may be an effective tool for detection of exposure to subtypes associated with highly pathogenic AIV. Our study provides further evidence of high seroprevalence in Arctic goose populations, and estimates of annual AIV seroconversion and antibody persistence for North American waterfowl. We suggest future AIV studies include serology to help elucidate the epizootiological dynamics of AIV in wild bird populations.

The potential use of ultrasonography (US) in evaluating gut disease has been underappreciated in most diagnostic imaging departments in North America. The impression that US has a questionable role in bowel assessment is related to the operator-dependent nature of the modality, the technical challenges of performing bowel US examinations, and the lack of familiarity of radiologists and technologists with the US appearances of normal and abnormal bowel. However, with development of technical experience by the sonographer and integration of a clinical focus at patient evaluation, US can become a powerful tool for bowel assessment. Unlike computed tomography and magnetic resonance imaging, it provides a widely available, noninvasive, inexpensive method for evaluating the gut without the use of ionizing radiation. These factors are of particular importance in young patients and those who require recurrent follow-up imaging. Because US is performed with real-time imaging, the modality also allows the sonographer to view and assess the motility properties of the bowel, a feature that has not been previously used to its full potential. Color Doppler US can yield useful information about mural vascularity in bowel disease when used in conjunction with gray-scale findings and clinical symptoms. Radiologists should be familiar with the static and dynamic US appearances of the normal and abnormal bowel, recognize features of various pathologic conditions, and understand potential errors at imaging interpretation. Online supplemental material is available for this article., (RSNA, 2015)

An effort aimed at exploring structural diversity in the N-pyrazole-N'-naphthylurea class of p38 kinase inhibitors led to the synthesis and characterization of N-phenyl-N'-naphthylureas. Examples of these compounds displayed excellent inhibition of TNF-alpha production in vitro, as well as efficacy in a mouse model of lipopolysaccharide induced endotoxemia. In addition, perspective is provided on the role of a sulfonamide functionality in defining inhibitor potency.

A computer-aided drug design strategy leads to the identification of a new class of p38 inhibitors based on the 2-tolyl-(1,2,3-triazol-1-yl-4-carboxamide) scaffold. The tolyl triazole amides provided a potent platform amenable to optimization. Further exploration leads to compounds with greater than 100-fold improvement in binding affinity to p38. Derivatives prepared to alter the physicochemical properties produced inhibitors with IC(50)'s in human whole blood as low as 83 nM.

Chemistry has been developed to specifically functionalize two structurally similar classes of indole-based MK2 inhibitors at positions prompted by a combination of X-ray crystallographic and computer assisted drug design. A gain in molecular potency was obtained by introducing aminomethyl groups to the lactam rings of 6-arylcarbamoyl-tetrahydro-beta-carbolinone and 6-arylcarbamoyl-dihydropyrazino[1,2-a]indolone MK2 inhibitors. In addition, improvements in molecular potency were achieved by expansion of the lactam from a 6- to 7-membered ring leading to 7-arylcarbamoyl-tetrahydro-[1,4]diazepino[1,2-a]indolones.

Optimization of pyrazinoindolone inhibitors of MAPKAP-K2 (MK2) provides a reasonable balance of cellular potency and physicochemical properties. Mechanistic studies support the inhibition of MK2 which is responsible for the sub-micromolar cellular efficacy.

Integration of computational methods, X-ray crystallography, and structure-activity relationships will be disclosed, which lead to a new class of p38 inhibitors that bind to p38 MAP kinase in a Phe out conformation.

The discovery of a series of potent, carboline-based MK2 inhibitors is described. These compounds inhibit MK2 with IC50s as low as 10 nM, as measured in a DELFIA assay. An X-ray crystal structure reveals that they bind in a region near the p-loop and the hinge region of MK2a.

Avian cholera, caused by Pasteurella multocida, affects waterbirds across North America and occurs worldwide among various avian species. Once an epizootic begins, contamination of the wetland environment likely facilitates the transmission of P. multocida to susceptible birds. To evaluate the ability of P. multocida serotype-1, the most common serotype associated with avian cholera in waterfowl in western and central North America, to persist in wetlands and to identify environmental factors associated with its persistence, we collected water and sediment samples from 23 wetlands during winters and springs of 1996-99. These samples were collected during avian cholera outbreaks and for up to 13 wk following initial sampling. We recovered P. multocida from six wetlands that were sampled following the initial outbreaks, but no P. multocida was isolated later than 7 wk after the initial outbreak sampling. We found no significant relationship between the probability of recovery of P. multocida during resampling and the abundance of the bacterium recovered during initial sampling, the substrate from which isolates were collected, isolate virulence, or water quality conditions previously suggested to be related to the abundance or survival of P. multocida. Our results indicate that wetlands are unlikely to serve as a long-term reservoir for P. multocida because the bacterium does not persist in wetlands for long time periods following avian cholera outbreaks.

We conducted a 3-yr study (2001-03) on greater white-fronted geese (Anser albifrons frontalis) breeding in Alaska, USA, to determine the exposure of this population to Pasteurella multocida and the potential role of these birds as disease carriers. We tested sera from nearly 600 adult geese for antibodies to P. multocida serotype 1. We found a low prevalence (<5%) of positive antibodies in adult geese, and based on the short duration of detectable antibodies, these findings indicate recent infection with P. multocida. Prevalence was similar to serologic results from both breeding and wintering lesser snow geese. We also collected oral (n=1,035), nasal (n=102), and cloacal (n=90) swab samples to determine the presence of avian cholera carriers in this population. We were unable to isolate P. multocida serotype 1 from any of the birds sampled. Based on comparison with other waterfowl species, we concluded that these geese may be exposed to avian cholera during the winter or spring migration but are unlikely to play a significant role as carriers of the bacterium causing avian cholera.

We collected samples from apparently healthy geese in the Playa Lakes Region (USA) during the winters of 2000-01 and 2001-02 to determine whether carriers of Pasteurella multocida, the bacterium that causes avian cholera, were present in wild populations. With the use of methods developed in laboratory challenge trials (Samuel et al., 2003a) and a serotype-specific polymerase chain reaction method for identification of P. multocida serotype 1, we found that a small proportion of 322 wild birds (<5%) were carriers of pathogenic P. multocida. On the basis of serology, an additional group of these birds (<10%) were survivors of recent avian cholera infection. Our results confirm the hypothesis that wild waterfowl are carriers of avian cholera and add support for the hypothesis that wild birds are a reservoir for this disease. In concert with other research, this work indicates that enzootic infection with avian cholera occurs in lesser snow goose (Chen caerulescens caerulescens) populations throughout their annual cycle. Although fewer Ross's geese (Chen rossii) were sampled, we also found these birds were carriers of P. multocida. Even in the absence of disease outbreaks, serologic evidence indicates that chronic disease transmission and recent infection are apparently occurring year-round in these highly gregarious birds and that a small portion of these populations are potential carriers with active infection.

A novel class of lymphocyte function-associated antigen-1 (LFA-1) inhibitors is described. Discovered during the process to improve the physicochemical and metabolic properties of BIRT377 (1, Figure 1), a previously reported hydantoin-based LFA-1 inhibitor, these compounds are characterized by an imidazole-based 5,5-bicyclic scaffold, the 1,3,3-trisubstituted 1H-imidazo[1,2-alpha]imidazol-2-one (i.e. structure 3). The structure-activity relationship (SAR) shows that electron-withdrawing groups at C5 on the imidazole ring benefit potency and that oxygen-containing functional groups attached to a C5-sulfonyl or sulfonamide group further improve potency. This latter gain in potency is attributed to the interaction(s) of the functionalized sulfonyl/sulfonamide groups with the protein, likely polar-polar in nature, as suggested by SAR data. X-ray studies revealed that these bicyclic inhibitors bind to the I-domain of LFA-1 in a pattern similar to that of compound 1.

Wetlands have long been suspected to be an important reservoir for Pasteurella multocida and therefore the likely source of avian cholera outbreaks. During the fall of 1995-98 we collected sediment and water samples from 44 wetlands where avian cholera epizootics occurred the previous winter or spring. We attempted to isolate P. multocida in sediment and surface water samples from 10 locations distributed throughout each wetland. We were not able to isolate P. multocida from any of the 440 water and 440 sediment samples collected from these wetlands. In contrast, during other investigations of avian cholera we isolated P. multocida from 20 of 44 wetlands, including 7% of the water and 4.5% of the sediment samples collected during or shortly following epizootic events. Our results indicate that wetlands are an unlikely reservoir for the bacteria that causes avian cholera.

We cultured 126 Pasteurella multocida isolates, 92 from water and 34 from sediment samples collected from wetlands in the Pacific and Central flyways of the United States between 1996 and 1999. Most (121) of the isolates were P. multocida serotype 1, but serotypes 3, 3/4, 10, and 11 were also found. Many (82) of the isolates were further characterized by DNA fingerprinting procedures and tested in Pekin ducks for virulence. Almost all the serotype 1 isolates we tested caused mortality in Pekin ducks. Serotype 1 isolates varied in virulence, but the most consistent pattern was higher mortality in male ducks than in females. We found no evidence that isolates found in sediment vs. water, between Pacific and Central flyways, or during El Niño years had consistently different virulence. We also found a number of non-serotype 1 isolates that were avirulent in Pekin ducks. Isolates had DNA fingerprint profiles similar to those found in birds that died during avian cholera outbreaks.

The discovery of novel 5,7-disubstituted[1,6]naphthyridines as potent inhibitors of Spleen Tyrosine Kinase (SYK) is discussed. The SAR reveals the necessity for a 7-aryl group with preference towards para substitution and that this in combination with 5-aminoalkylamino substituents further improved the potency of the compounds. The initial SAR as well as a survey of the other positions is discussed in detail.

The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors based on the phenylaminoimidazoisoquinolin-9-one showed reasonable cellular activity but did not work in vivo upon oral administration. Our current work highlights the further use of rational drug design and molecular modeling to produce a series of lck inhibitors that demonstrate cellular activity below 100 nM and are as efficacious as cyclosporin A in an in vivo mouse model of anti-CD3-induced IL-2 production.

We conducted laboratory challenge trials using mallard ducks (Anas platyrhynchos) to compare methods for detecting carriers of Pasteurella multocida, the bacterium that causes avian cholera, in wild birds. Birds that survived the initial infection were euthanized at 2-4 wk intervals up to 14 wk post challenge. Isolates of P. multocida were obtained at necropsy from 23% of the birds that survived initial infection. We found that swab samples (oral, cloacal, nasal, eye, and leg joint) were most effective for detecting carrier birds up to 14 wk post infection. No detectable differences in isolation were observed for samples stored in either 10% dimethysulfoxide or brain heart infusion broth. The frequency of detecting carriers in our challenge trials appeared to be related to mortality rates observed during the trial, but was not related to a number of other factors including time after challenge, time delays in collecting tissues postmortem, and route of infection. In our trials, there was little association between antibody levels and carrier status. We concluded that swabs samples collected from recently dead birds, stored in liquid nitrogen, and processed using selective broth provide a feasible field method for detecting P. multocida carriers in wild waterfowl.

To determine if lesser snow geese (Chen caerulescens caerulescens) are a potential reservoir for the Pasteurella multocida bacterium that causes avian cholera, serum samples and/or pharyngeal swabs were collected from > 3,400 adult geese breeding on Wrangel Island (Russia) and Banks Island (Canada) during 1993-1996. Pharyngeal swab sampling rarely (> 0.1%) detected birds that were exposed to P. multocida in these populations. Geese with serum antibody levels indicating recent infection with P. multocida were found at both breeding colonies. Prevalence of seropositive birds was 3.5% at Wrangel Island, an area that has no recorded history of avian cholera epizootics. Prevalence of seropositive birds was 2.8% at Banks Island in 1994, but increased to 8.2% during 1995 and 1996 when an estimated 40,000-60,000 snow geese were infected. Approximately 50% of the infected birds died during the epizootic and a portion of the surviving birds may have become carriers of the disease. This pattern of prevalence indicated that enzootic levels of infection with P. multocida occurred at both breeding colonies. When no avian cholera epizootics occurred (Wrangel Island, Banks Island in 1994), female snow geese (4.7%) had higher antibody prevalence than males (2.0%).

A cryopreservation method and improved isolation techniques for detection of Pasteurella multocida from wetland samples were developed. Wetland water samples were collected in the field, diluted in dimethyl sulfoxide (DMSO, final concentration 10%), and frozen at -180 C in a liquid nitrogen vapor shipper. Frozen samples were transported to the laboratory where they were subsequently thawed and processed in Pasteurella multocida selective broth (PMSB) to isolate P. multocida. This method allowed for consistent isolation of 2 to 18 organisms/ml from water seeded with known concentrations of P. multocida. The method compared favorably with the standard mouse inoculation method and allowed for preservation of the samples until they could be processed in the laboratory.

Pharyngeal swabs were collected from 298 lesser snow geese (Chen caerulescens caerulescens) at Banks Island (Northwest Territories. Canada) in the summer of 1994. Pasteurella multocida serotype 1 was isolated from an adult male bird and P. multocida serotype 3 was isolated from an adult female goose. Pathogenicity of the serotype 1 isolate was confirmed by inoculation in Pekin ducks (Anas platyrhynchos). The serotype 3 isolate was non-pathogenic in Pekin ducks. This is the first documented isolation of pathogenic P. multocida serotype 1 from apparently healthy wild snow geese.

We developed an ELISA procedure to assess the presence of M. anatis-specific serum antibody in ducks. Sera from exposed and unexposed Pekin ducks (Anas platyrhynchos) were used to standardize the ELISA and to establish reference ranges to classify ELISA results as exposed or not exposed. We conducted serological surveys of female waterfowl in the central and eastern United States between 1988 and 1992 to assess the frequency of exposure in wild waterfowl. Adult breeding mallards (Anas platyrhynchos), wintering mallards, and black ducks (Anas rubripes) had high prevalences of exposure to M. anatis (25% to > 80%). In comparison, none of the breeding adult canvasbacks (Aythya valisineria) had serum antibody levels indicating exposure. Approximately 50% of the juvenile mallards and black ducks were exposed to M. anatis by 8 months of age, indicating high transmission rates among wild birds.

We determined the prevalence of mycoplasma infection in breeding mallard (Anas platyrhynchos) and canvasback (Aythya valisineria) hens and their broods from the central United States (1988 to 1990); and wintering American black duck (Anas rubripes) and mallard hens from the eastern United States (1990 to 1993). Mycoplasmas were isolated by culturing tracheal swabs from 656 live birds and tissue samples from 112 dead waterfowl. Nine (18%) of 51 mycoplasma isolates were identified as Mycoplasma anatis; M. anatis was recovered from four mallards, a black duck, and a gadwall (Anas strepera) duckling. Nineteen (37%) of 51 mycoplasma isolates were identified as Mycoplasma cloacale; these isolates were obtained from mallard, canvasback, and black duck adults, and from a mallard duckling. Additional unspeciated mycoplasmas were isolated from mallards, black ducks, and one canvasback.

We inoculated game-farm mallard (Anas platyrhynchos) eggs and 1-day-old birds with Mycoplasma anatis to determine its effect on hatching success and growth rates of ducklings. Inoculations of eggs reduced hatching success, hatchling size, and duckling growth rates, compared to controls. Intratracheal inoculations of 1-day-old birds did not affect growth rates. Hatchlings and 1-day-old ducklings grew much slower for the first 7 to 10 days when raised at 17 to 19 C, compared to controls raised at 30 to 35 C. The effect of cold stress on growth was greater than the effect of M. anatis infection; we found no synergistic effects between cold stress and M. anatis infection.

Environmental contaminants contain chemicals that, if ingested, could affect the immunological status of wild birds, and in particular, their resistance to infectious disease. Immunosuppression caused by environmental contaminants, could have a major impact on waterfowl populations, resulting in increased susceptibility to contagious disease agents. Duck plague virus has caused repeated outbreaks in waterfowl resulting in mortality. In this study, several doses of cyclophosphamide (CY), a known immunosuppressant, were administered to adult mallards (Anas platyrhynchos) to determine if a resultant decrease in resistance to a normally sub-lethal strain of duck plague virus would occur, and induce mortality in these birds. Death occurred in birds given CY only, and in birds given virus and CY, but not in those given virus only. There was significantly greater mortality and more rapid deaths in the duck plague virus-infected groups than in groups receiving only the immunosuppressant. A positively correlated dose-response effect was observed with CY mortalities, irrespective of virus exposure. A fuel oil and a crude oil, common environmental contaminants with immunosuppressive capabilities, were tested to determine if they could produce an effect similar to that of CY. Following 28 days of oral oil administration, the birds were challenged with a sub-lethal dose of duck plague virus. No alteration in resistance to the virus (as measured by mortality) was observed, except in the positive CY control group.

Sewage sludge contains numerous chemicals that, if ingested, could affect the immunological status of wild birds and, in particular, their resistance to infectious disease. Mallard ducks were fed a diet containing 0, 5, or 20% dried sewage sludge from either Milwaukee or Madison, Wisconsin, for 28 and 57 days, respectively. When subsequently challenged with Pasteurella multocida, the mortality in the sludge-treated groups was no greater than that in the untreated control groups. There was, however, significantly more cadmium (two- to three-fold higher concentration) retained in the livers of birds receiving 20% of either sludge in their feed, as compared to controls.