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Background: Published evidence indicates that mean arterial pressure (MAP) below a goal range (hypotension) is associated with worse outcomes, though MAP management failures are common. We sought to characterize hypotension occurrences in ICUs and consider the implications for MAP management. Methods: Retrospective analysis of 3 hospitals' cohorts of adult ICU patients during continuous vasopressor infusion. Two cohorts were general, mixed ICU patients and one was exclusively acute spinal cord injury patients. "Hypotension-clusters" were defined where there were ≥10 min of cumulative hypotension over a 60-min period and "constant hypotension" was ≥10 continuous minutes. Trend analysis was performed (predicting future MAP using 14 min of preceding MAP data) to understand which hypotension-clusters could likely have been predicted by clinician awareness of MAP trends. Results: In cohorts of 155, 66, and 16 ICU stays, respectively, the majority of hypotension occurred within the hypotension-clusters. Failures to keep MAP above the hypotension threshold were notable in the bottom quartiles of each cohort, with hypotension durations of 436, 167, and 468 min, respectively, occurring within hypotension-clusters per day. Mean arterial pressure trend analysis identified most hypotension-clusters before any constant hypotension occurred (81.2%-93.6% sensitivity, range). The positive predictive value of hypotension predictions ranged from 51.4% to 72.9%. Conclusions: Across 3 cohorts, most hypotension occurred in temporal clusters of hypotension that were usually predictable from extrapolation of MAP trends., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Supported by and performed in collaboration with Nihon Kohden Corporation and the Nihon Kohden Innovation Center.

Here, we provide evidence that the freshwater parasitic copepod, Salmincola californiensis, acts as a vector for Aeromonas salmonicida. While investigating the effects of S. californiensis on Chinoook salmon (Oncorhynchus tshawytscha), we tangentially observed that fish infected with the copepod developed furunculosis, caused by A. salmonicida. This occurred despite being reared in pathogen-free well water in a research facility with no prior history of spontaneous infection. We further investigated the possibility of S. californiensis to serve as a vector for the bacterium via detection of fluorescently labelled A. salmonicida inside the egg sacs from copepods in which the fish hosts were experimentally infected with GFP-A449 A. salmonicida. We then evaluated copepod egg sacs that were collected from adult Chinook salmon from a freshwater hatchery with A. salmonicida infections confirmed by either culture or PCR. The bacterium was cultured on tryptic soy agar plates from 75% of the egg sacs, and 61% were positive by PCR. These three separate experiments indicate an alternative tactic of transmission in addition to direct transmission of A. salmonicida in captivity. The copepod may play an important role in transmission of the bacterium when fish are more dispersed, such as in the wild., (© 2023 John Wiley & Sons Ltd.)

Prespawn mortality (PSM) presents a major problem for the recovery of spring Chinook Salmon (Oncorhynchus tshawytscha) populations. In the Willamette River, Oregon, PSM exceeds 90% in some years but factors explaining it are not well understood. We examined intestinal tissue samples using histological slides from over 783 spring Chinook Salmon collected between 2009 and 2021, which included tissues from PSM fish, artificially spawned captive broodstock (BS) and normal river run fish, comprised of trapped (Live) and naturally post-spawned river (RPS) fish collected from the river. We observed degeneration of the intestinal epithelium and loss of villous structure, with concurrent severe enteritis. A natural progression of decline in epithelial integrity (EI) through the summer and fall until spawning and subsequent death was also observed. Live fish exhibited high EI scores (mean = 68%), BS exhibited variable EI scores (35%) and RPS exhibited severe loss of EI (14%). PSM fish exhibited prominent loss of intestinal epithelium with EI scores (13%), very similar to RPS fish, despite having been collected earlier in the year. Hence, we argue that low EI scores are strongly linked with PSM. Ceratonova shasta and Enterocytozoon schreckii were common in all groups, but neither were linked to either PSM or a decline in EI., (© 2023 John Wiley & Sons Ltd.)

Background and Purpose: An increased number of pathogenic variants have been described in mitochondrial encephalomyopathy lactic acidosis and strokelike episodes (MELAS). Different imaging presentations have emerged in parallel with a growing recognition of clinical and outcome variability, which pose a diagnostic challenge to neurologists and radiologists and may impact an individual patient's response to therapeutic interventions. By evaluating clinical, neuroimaging, laboratory, and genetic findings, we sought to improve our understanding of the sources of potential phenotype variability in patients with MELAS., Materials and Methods: This retrospective single-center study included individuals who had confirmed mitochondrial DNA pathogenic variants and a diagnosis of MELAS and whose data were reviewed from January 2000 through November 2021. The approach included a review of clinical, neuroimaging, laboratory, and genetic data, followed by an unsupervised hierarchical cluster analysis looking for sources of phenotype variability in MELAS. Subsequently, experts identified "victory-variables" that best differentiated MELAS cohort clusters., Results: Thirty-five patients with a diagnosis of mitochondrial DNA-based MELAS (median age, 12 years; interquartile range, 7-24 years; 24 female) were eligible for this study. Fifty-three discrete variables were evaluated by an unsupervised cluster analysis, which revealed that two distinct phenotypes exist among patients with MELAS. After experts reviewed the variables, they selected 8 victory-variables with the greatest impact in determining the MELAS subgroups: developmental delay, sensorineural hearing loss, vision loss in the first strokelike episode, Leigh syndrome overlap, age at the first strokelike episode, cortical lesion size, regional brain distribution of lesions, and genetic groups. Ultimately, 2-step differentiating criteria were defined to classify atypical MELAS., Conclusions: We identified 2 distinct patterns of MELAS: classic MELAS and atypical MELAS. Recognizing different patterns in MELAS presentations will enable clinical and research care teams to better understand the natural history and prognosis of MELAS and identify the best candidates for specific therapeutic interventions., (© 2023 by American Journal of Neuroradiology.)

Pacific salmon experience prolonged elevation in corticosteroid hormones during important life history events including migration, reproduction, and senescence. These periods of elevated corticosteroids correspond with changes to immunity and energy metabolism; therefore, fish may be particularly vulnerable to mortality at these times. Recent studies found that stress-induced cortisol release associated with microbial community shifts in salmonids, raising the question of how longer-term corticosteroid dynamics that accompany life history transitions affect salmonid microbiomes. In this work, we experimentally evaluated the relationships between gut microbiome composition, chronically elevated corticosteroids, and mortality in juvenile Chinook salmon (Oncorhynchus tshawytscha). We found that treatment with slow-release implants of the corticosteroids cortisol or dexamethasone resulted in changes to the gut microbiome. Morbidity was also associated with microbiome composition, suggesting that the gut microbiome reflects individual differences in susceptibility to opportunistic pathogens. Additionally, we analyzed a small number of samples from adult fish at various stages of senescence. Results from these samples suggest that microbiome composition associated with gut integrity, and that the microbial communities of corticosteroid treated juveniles shift in composition toward those of senescent adults. Overall, findings from this work indicate that the gut microbiome correlates with mortality risk during periods of chronic corticosteroid elevation., (© 2023. The Author(s).)

Detecting the presence of important parasites within a host and its environment is critical to understanding the dynamics that influence a pathogen's ability to persist, while accurate detection is also essential for the implementation of effective control strategies. Pseudoloma neurophilia is the most common pathogen reported in zebrafish (Danio rerio) research facilities. The only assays currently available for P. neurophilia are through lethal sampling, often requiring euthanasia of the entire population for accurate estimates of prevalence in small populations. We present a non-lethal screening method to detect P. neurophilia in tank water based on the detection of environmental DNA (eDNA) from this microsporidium, using a previously developed qPCR assay that was adapted to the digital PCR (dPCR) platform to complement current surveillance protocols. Using the generated dPCR data, a multi-state occupancy model was also implemented to predict the probability of detecting the microsporidium in tank water under different flow regimes and pathogen prevalence. The occupancy model revealed that samples collected in static conditions were more informative than samples collected from flow-through conditions, with a probability of detection at 80% and 47%, respectively. There was also a positive correlation between the frequency of detection in water and prevalence in fish based on qPCR., (© American Society of Parasitologists 2022.)

Pseudoloma neurophilia is a critical threat to the zebrafish (Danio rerio) model, as it is the most common infectious agent found in research facilities. In this study, our objectives were two-fold: (1) compare the application of diagnostic tools for P. neurophilia and (2) track the progression of infection using PCR and histology. The first experiment showed that whole-body analysis by qPCR (WB-qPCR) can be a standardized process, providing a streamlined diagnostic protocol, without the need for extraction of specific tissues. Evaluating the course of infection in experimentally infected fish, we showed key dynamics in infection. Starting with a low dose exposure of 8000 spores/fish, the prevalence remained low until 92 days post-exposure (dpe), followed by a 30%-40% prevalence by histology or 40%-90% by PCR until the end of the experiment at 334 dpe. WB-qPCR positively detected infection in more fish than histology throughout the study, as WB-qPCR detected the parasite as early as 4 dpe, whereas it was undetected by histology until 92 dpe. We also added a second slide for histologic analyses, showing an increase in detection rate from 24% to 26% when we combined all data from our experiments, but this increase was not statistically significant., (© 2022 John Wiley & Sons Ltd.)

Background: Prevention of respiratory syncytial virus (RSV) disease in infants is an unmet vaccine need, and maternal immunization is a potential strategy to address this need. This study evaluated concomitant administration of RSV stabilized prefusion F subunit vaccine (RSVpreF) and tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine adsorbed (Tdap) in healthy, nonpregnant women 18‒49 years of age., Methods: In this phase 2b, multicenter, placebo-controlled, observer-blind, noninferiority study, participants were randomized to receive RSVpreF in a range of doses and formulations with Tdap or alone, or Tdap alone. Safety and immunogenicity were assessed., Results: Local reactions and systemic events were generally similar across vaccine groups. Noninferiority of anti-RSV-A and anti-RSV-B immune responses induced by RSVpreF with Tdap was demonstrated compared to RSVpreF alone. Noninferiority of anti-diphtheria toxoid and anti-tetanus toxoid immune responses after administration of RSVpreF with Tdap was demonstrated compared to Tdap alone; noninferiority was not met for anti-pertussis component responses., Conclusions: RSVpreF was safe and well tolerated when administered with Tdap or alone in nonpregnant women 18‒49 years of age. Immune responses induced by Tdap administered with RSVpreF were noninferior for the tetanus and diphtheria components of Tdap, but not for pertussis., Clinical Trials Registration: NCT04071158., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

Mitochondrial disease diagnosis requires interrogation of both nuclear and mitochondrial (mtDNA) genomes for single-nucleotide variants (SNVs) and copy number alterations, both in the proband and often maternal relatives, together with careful phenotype correlation. We developed a comprehensive mtDNA sequencing test ('MitoGenome') using long-range PCR (LR-PCR) to amplify the full length of the mtDNA genome followed by next generation sequencing (NGS) to accurately detect SNVs and large-scale mtDNA deletions (LSMD), combined with droplet digital PCR (ddPCR) for LSMD heteroplasmy quantification. Overall, MitoGenome tests were performed on 428 samples from 394 patients with suspected or confirmed mitochondrial disease. The positive yield was 11% (43/394), including 34 patients with pathogenic or likely pathogenic SNVs (the most common being m.3243A > G in 8/34 (24%) patients), 8 patients with single LSMD, and 3 patients with multiple LSMD exceeding 10% heteroplasmy levels. Two patients with both LSMD and pathogenic SNV were detected. Overall, this LR-PCR/NGS assay provides a highly accurate and comprehensive diagnostic method for simultaneous mtDNA SNV detection at heteroplasmy levels as low as 1% and LSMD detection at heteroplasmy levels below 10%. Inclusion of maternal samples for variant classification and ddPCR to quantify LSMD heteroplasmy levels further enables accurate pathogenicity assessment and clinical correlation interpretation of mtDNA genome sequence variants and copy number alterations., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Occupancy models are often used to analyze long-term monitoring data to better understand how and why species redistribute across dynamic landscapes while accounting for incomplete capture. However, this approach requires replicate detection/non-detection data at a sample unit and many long-term monitoring programs lack temporal replicate surveys. In such cases, it has been suggested that surveying subunits within a larger sample unit may be an efficient substitution (i.e., space-for-time substitution). Still, the efficacy of fitting occupancy models using a space-for-time substitution has not been fully explored and is likely context dependent. Herein, we fit occupancy models to Delta Smelt ( Hypomesus transpacificus ) and Longfin Smelt ( Spirinchus thaleichthys ) catch data collected by two different monitoring programs that use the same sampling gear in the San Francisco Bay-Delta, USA. We demonstrate how our inferences concerning the distribution of these species changes when using a space-for-time substitution. Specifically, we found the probability that a sample unit was occupied was much greater when using a space-for-time substitution, presumably due to the change in the spatial scale of our inferences. Furthermore, we observed that as the spatial scale of our inferences increased, our ability to detect environmental effects on system dynamics was obscured, which we suspect is related to the tradeoffs associated with spatial grain and extent. Overall, our findings highlight the importance of considering how the unique characteristics of monitoring programs influences inferences, which has broad implications for how to appropriately leverage existing long-term monitoring data to understand the distribution of species., Competing Interests: None declared., (Published 2021. This article is a U.S.Government work and is in the public domain in the USA. Ecology and Evolution published by John Wiley & Sons Ltd.)

Pacific salmon (Oncorhynchus spp.) rearing in lakes and reservoirs above dams have been known to become heavily infected with an ectoparasitic copepod (Salmincola californiensis). Little is known about the factors that affect the parasite infection prevalence and intensity. However, previous research suggests that the parasite may negatively affect the fitness and survival of the host fish. The effect of water temperature, confinement and the density of the free-swimming infectious stage of S. californiensis, the copepodid, on infection prevalence and intensity was evaluated by experimentally exposing juvenile Chinook Salmon (O. tshawytscha). Infection rates observed in wild populations were achieved under warm water (15-16°C) and high copepodid density (150-300/L) treatment conditions. Infection prevalence and intensity were also significantly higher in larger fish. During the infection experiment, 4.5% of infected fish died within 54 days with mortality significantly related to copepod infection intensity. The potential for autoinfection was compared to cross-infection by cohabitation of infected fish with naïve fish. Previously infected fish had significantly greater infection intensity compared with naïve fish, indicating that infected fish can be reinfected and that they may be more susceptible than naïve fish., (© 2021 John Wiley & Sons Ltd. This article is a U.S. Government work and is in the public domain in the USA.)

In most countries worldwide, pneumococcal conjugate vaccines have been included in the infant immunization program, resulting in a significant reduction in the burden of pneumococcal disease in children and adults. Shifting serotype distribution due to the indirect effect of infant vaccination with the 13-valent pneumococcal conjugate vaccine (PCV13) may continue to increase the gap between 23-valent pneumococcal polysaccharide vaccine (PPSV23) and PCV13 serotype coverage for older adults in the coming years. This clinical study (V110-029; NCT02225587) evaluated the safety and immunogenicity of sequential administration of PCV13 followed approximately 8 weeks later, or approximately 26 weeks later, by PPSV23 in healthy adults ≥50 years of age. Both dosing intervals were generally well tolerated as measured by the nature, frequency, and intensity of reported adverse events (AEs) in both vaccination groups. Serotype-specific opsonophagocytic activity (OPA) geometric mean titers (GMTs) measured 30 days following receipt of PPSV23 in either group and at Week 30 were generally comparable between the 2 groups for 6 serotypes unique to PPSV23 and 12 serotypes shared between PCV13 and PPSV23, regardless of the interval between receipt of PCV13 and PPSV23. In addition, administration of PPSV23 given either 8 weeks or 26 weeks following PCV13 did not negatively impact immune responses induced by PCV13. Furthermore, administration of PPSV23 given either 8 weeks or 26 weeks after PCV13 elicited serotype-specific OPA GMTs to serotypes unique to PPSV23, which could provide earlier protection against pneumococcal disease caused by these serotypes in comparison with the current Advisory Committee on Immunization Practices recommended interval of at least 12 months.

Human 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) is a putative iron-containing non-heme oxygenase of unknown specificity and biological significance. We report 25 families containing 34 individuals with neurological disease associated with biallelic HPDL variants. Phenotypes ranged from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spasticity and global developmental delays, sometimes complicated by episodes of neurological and respiratory decompensation. Variants included bona fide pathogenic truncating changes, although most were missense substitutions. Functionality of variants could not be determined directly as the enzymatic specificity of HPDL is unknown; however, when HPDL missense substitutions were introduced into 4-hydroxyphenylpyruvate dioxygenase (HPPD, an HPDL orthologue), they impaired the ability of HPPD to convert 4-hydroxyphenylpyruvate into homogentisate. Moreover, three additional sets of experiments provided evidence for a role of HPDL in the nervous system and further supported its link to neurological disease: (i) HPDL was expressed in the nervous system and expression increased during neural differentiation; (ii) knockdown of zebrafish hpdl led to abnormal motor behaviour, replicating aspects of the human disease; and (iii) HPDL localized to mitochondria, consistent with mitochondrial disease that is often associated with neurological manifestations. Our findings suggest that biallelic HPDL variants cause a syndrome varying from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spastic tetraplegia associated with global developmental delays., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Bull trout (Salvelinus confluentus) are challenging to detect as a result of the species cryptic behavior and coloration, relatively low densities in complex habitats, and affinity for cold, high clarity, low conductivity waters. Bull trout are also closely associated with the stream bed, frequently conceal in substrate, and this concealment behavior is poorly understood. Consequently, population assessments are problematic and biologists and managers often lack quantitative information to accurately describe bull trout distributions, estimate abundance, and assess status and trends; particularly for stream-dwelling populations. During controlled laboratory trials, we recorded concealment, resting, and swimming behavior of juvenile wild bull trout in response to: (1) constant and fluctuating water temperature, (2) presence or absence of light, and (3) substrate size. Light level had the strongest influence on wild fish concealment and more fish concealed as light levels increased from darkness to daylight. Wild fish were 14.5 times less likely to conceal in constant darkness and 4.1 times more likely to conceal in 12 h light x 12 h darkness compared to constant light. Wild fish were 6.2 times less likely to conceal in small (26-51 mm) substrate compared to larger (52-102 mm) substrate. As water temperature increased, fewer wild fish concealed. Knowledge of wild bull trout concealment will improve field sampling protocols and increase detection efficiencies. These data also enhance knowledge of bull trout niche requirements which illuminates ecological differences among species and informs conservation and restoration efforts., Competing Interests: The authors have declared that no competing interests exist.

We investigated whether a statistical model could predict mean arterial pressure (MAP) during uncontrolled hemorrhage; such a model could be used for automated decision support, to help clinicians decide when to provide intravascular volume to achieve MAP goals. This was a secondary analysis of adult swine subjects during uncontrolled splenic bleeding. By protocol, after developing severe hypotension (MAP < 60 mmHg), subjects were resuscitated with either saline (NS) or fresh frozen plasma (FFP), determined randomly. Vital signs were documented at quasi-regular time-step intervals, until either subject death or 300 min. Subjects were randomly separated 50%/50% into training/validation sets, and regression models were developed to predict MAP for each subsequent (i.e., future) time-step. Median time-steps for serially recorded vital signs were +15 min. 5 subjects survived the protocol; 17 died after a median time of 87 min (IQR 78 - 134). The final model consisted of: current MAP; heart rate (HR); prior NS; imminent NS; and imminent FFP. The 95% limits-of-agreement between true subsequent MAP vs. predicted subsequent MAP were +10/-11 mmHg for the 79 time-steps in the training set; and +14/-13 for the 64 time-steps in the validation set. A total of 10 sudden death events (i.e., rapid, fatal MAP decrease within one single time-step) were excluded from analysis. In conclusion, for uncontrolled hemorrhage in a swine model, it was possible to estimate the next documented MAP value on the basis of the subject's current documented MAP; HR; prior NS; and the volume of resuscitation about to be administered. However, the model was unable to predict "sudden death" events. The applicability to populations with wider heterogeneity of hemorrhage patterns and with comorbidities requires further investigation.

We used odds ratios and a hurdle model to analyze parasite co-infections over 25 years on >20,000 young-of-the year of endangered Shortnose and Lost River Suckers. Host ecologies differed as did parasite infections. Shortnose Suckers were more likely to be caught inshore and 3-5 times more likely to have Bolbophorus spp. and Contracaecum sp. infections, and Lost River Suckers were more likely to be caught offshore and approximately three times more likely to have Lernaea cyprinacea infections. An observed peak shift seems likely to be due to a lower host size limit for Bolbophorus spp. (13.6 mm) compared with L. cyprinacea (23.4 mm). The large data set allowed us to generate strong hypotheses: (i) that a major marsh restoration project had unintended consequences that resulted in an increase in infections; (ii) that co-infection with Bolbophorus spp. increased the odds of infection by L. cyprinacea and Contracaecum sp.; (iii) that significant declines in the odds of infection over approximately 25 days were due to parasite-induced host mortality; (iv) that the fish's small size relative to L. cyprinacea and Contracaecum sp. might be directly lethal; (v) that the absence of L. cyprinacea infections in the early 1990s was associated with good year-class production of the suckers; and (vi) that parasites might increase the odds of vagrancy from the nursery ground., (Copyright © 2020 Australian Society for Parasitology. All rights reserved.)

Background: Pneumococcal disease remains a public health priority in adults. Previous studies have suggested that administration of pneumococcal polysaccharide vaccine or pneumococcal conjugate vaccine within three years following receipt of PPV23 was associated with increased reactogenicity and reduced antibody titers in comparison to longer intervals. Safety and immunogenicity of 15-valent pneumococcal conjugate vaccine (PCV15) was evaluated in adults ≥ 65 years of age with prior history of PPV23 vaccination (V114-007; NCT02573181)., Methods: A total of 250 adults who received PPV23 at least 1 year prior to study entry received a single dose of either PCV15 or PCV13 (125/arm) and were followed for safety for 14 days postvaccination. Serotype-specific Immunoglobulin G (IgG) geometric mean concentrations (GMCs) and opsonophagocytic activity (OPA) geometric mean titers (GMTs) were measured immediately prior and 30 days postvaccination., Results: Safety profiles were comparable between PCV15 and PCV13 recipients. Following vaccination, serotype-specific antibody responses for the 13 shared serotypes were generally comparable between recipients of PCV15 and PCV13 for IgG GMCs, OPA GMTs, and geometric mean fold rises (GMFRs) and percentages of subjects with ≥ 4-fold-rise from baseline for both IgG and OPA. Recipients of PCV15 had numerically higher antibody responses than PCV13 for two serotypes unique to PCV15 (22F, 33F)., Conclusion: PCV15 was generally well tolerated and induced high levels of IgG and OPA antibodies to all 15 serotypes included in the vaccine when given as a single dose to adults ≥ 65 years of age previously vaccinated with PPV23.

Background: Pneumococcal disease remains a public health priority in adults. Safety and immunogenicity of 2 different formulations of 15-valent pneumococcal conjugate vaccine (PCV15) containing 13 serotypes included in 13-valent pneumococcal conjugate vaccine (PCV13) plus 2 additional serotypes (22F and 33F) were evaluated in adults ≥ 50 years (V114-006; NCT02547649)., Methods: A total of 690 subjects (230/arm) received a single dose of either PCV15 Formulation A, PCV15 Formulation B, or PCV13 and were followed for safety for 14 days postvaccination. Serotype-specific opsonophagocytic activity (OPA) geometric mean titers (GMTs) and Immunoglobulin G (IgG) geometric mean concentrations (GMCs) were measured immediately prior and 30 days postvaccination., Results: Both PCV15 formulations had generally comparable safety profiles to PCV13. Baseline IgG GMCs and OPA GMTs were comparable across vaccination groups. At 30 days postvaccination, both PCV15 formulations induced serotype specific antibodies to all 15 serotypes in the vaccine. IgG GMCs and OPA GMTs in recipients of either PCV15 formulation were non-inferior (≤ 2-fold margin) to those measured in recipients of PCV13 for shared serotypes and superior (> 1.0-fold difference) for serotypes unique to PCV15. Formulation B generally induced higher immune responses than Formulation A., Conclusion: In healthy adults ≥ 50 years of age, both new formulations of PCV15 displayed acceptable safety profiles and induced serotype-specific immune responses comparable to PCV13.

Introduction: Porters working at high altitude face a number of preventable health risks ranging from frostbite to potentially fatal high-altitude pulmonary and cerebral edema. Porters are often recruited from impoverished low-altitude areas, poorly equipped in terms of protective clothing, and tasked with carrying loads that equal or exceed their body mass to high elevations. Despite a large population of porters working throughout mountainous parts of the world, there is little documentation regarding knowledge levels, attitudes, and practices related to the prevention of altitude illness in this population. Much of the literature focuses on biomechanics of load carrying for porters or altitude issues for trekkers., Methods: A cross-sectional survey was developed using a modified Delphi technique. The survey was administered to a convenience sample of porters (n=146) from diverse cultural groups between Lukla (2774 m) and Everest Base Camp (5361 m)., Results: Many of the porters started at a young age, carried heavy loads, and had difficulty identifying symptoms of high altitude illness, and less than 20% correctly identified preventive measures for high-altitude illness., Conclusions: Porters in the Khumbu region continue to face hazards in their working environment. Future research and health education campaigns should address knowledge deficits and help with the design of tailored interventions., (Copyright © 2018 Wilderness Medical Society. Published by Elsevier Inc. All rights reserved.)

Managers are increasingly implementing reintroduction programs as part of a global effort to alleviate amphibian declines. Given uncertainty in factors affecting populations and a need to make recurring decisions to achieve objectives, adaptive management is a useful component of these efforts. A major impediment to the estimation of demographic rates often used to parameterize and refine decision-support models is that life-stage-specific monitoring data are frequently sparse for amphibians. We developed a new parameterization for integrated population models to match the ecology of amphibians and capitalize on relatively inexpensive monitoring data to document amphibian reintroductions. We evaluate the capability of this model by fitting it to Oregon spotted frog (Rana pretiosa) monitoring data collected from 2007 to 2014 following their reintroduction within the Klamath Basin, Oregon, USA. The number of egg masses encountered and the estimated adult and metamorph abundances generally increased following reintroduction. We found that survival probability from egg to metamorph ranged from 0.01 in 2008 to 0.09 in 2009 and was not related to minimum spring temperatures, metamorph survival probability ranged from 0.13 in 2010-2011 to 0.86 in 2012-2013 and was positively related to mean monthly temperatures (logit-scale slope = 2.37), adult survival probability was lower for founders (0.40) than individuals recruited after reintroduction (0.56), and the mean number of egg masses per adult female was 0.74. Our study is the first to test hypotheses concerning Oregon spotted frog egg-to-metamorph and metamorph-to-adult transition probabilities in the wild and document their response at multiple life stages following reintroduction. Furthermore, we provide an example to illustrate how the structure of our integrated population model serves as a useful foundation for amphibian decision-support models within adaptive management programs. The integration of multiple, but related, data sets has an advantage of being able to estimate complex ecological relationships across multiple life stages, offering a modeling framework that accommodates uncertainty, enforces parsimony, and ensures all model parameters can be confronted with monitoring data., (© 2017 by the Ecological Society of America.)

Stream ecosystems provide multiple, valued services to society, including water supply, waste assimilation, recreation, and habitat for diverse and productive biological communities. Managers striving to sustain these services in the face of changing climate, land uses, and water demands need tools to assess the potential effectiveness of alternative management actions, and often, the resulting tradeoffs between competing objectives. Integrating predictive modeling with monitoring data in an adaptive management framework provides a process by which managers can reduce model uncertainties and thus improve the scientific bases for subsequent decisions. We demonstrate an integration of monitoring data with a dynamic, metapopulation model developed to assess effects of streamflow alteration on fish occupancy in a southeastern US stream system. Although not extensive (collected over three years at nine sites), the monitoring data allowed us to assess and update support for alternative population dynamic models using model probabilities and Bayes rule. We then use the updated model weights to estimate the effects of water withdrawal on stream fish communities and demonstrate how feedback in the form of monitoring data can be used to improve water resource decision making. We conclude that investment in more strategic monitoring, guided by a priori model predictions under alternative hypotheses and an adaptive sampling design, could substantially improve the information available to guide decision-making and management for ecosystem services from lotic systems., (Published by Elsevier Ltd.)

Objective: Patients with diabetes mellitus are at increased risk for hepatitis B virus (HBV) infection and its complications. HBV vaccination is recommended for adults with diabetes in the United States and other countries. However, few studies have assessed safety and immunogenicity of hepatitis B vaccine in such patients. We assessed the safety and immunogenicity of recombinant hepatitis B vaccine in subjects with and without diabetes mellitus., Methods: Prospective, multi-country controlled study in 21 centers ( www.clinicaltrials.gov NCT01627340). Four hundred and sixteen participants with Type-2 diabetes and 258 controls matched for age and body mass index (BMI) (2:1 ratio) received 3-doses of HBV vaccine (Engerix-B™, GSK Vaccines, Belgium) according to a 0, 1, 6 months schedule. Antibodies were measured against HBV surface antigen and expressed as seroprotection rates (anti-HBs ≥10mIU/mL) and geometric mean concentration (GMC)., Results: The median age and BMI in patients with diabetes and controls (according-to-protocol cohort) were 54 y and 32.1 kg/m 2 , and 53 y and 30.8 kg/m 2 , respectively. Seroprotection rates (GMCs) one month post-dose-3 were 75.4% (147.6 mIU/mL) and 82.0% (384.2 mIU/mL) in patients with diabetes and controls, respectively. Age-stratified seroprotection rates for patients with diabetes were 88.5% (20-39 years), 81.2% (40-49 years), 83.2% (50-59 years), and 58.2% (≥60 years). The overall safety profile of hepatitis B vaccine was similar between groups., Conclusions: Hepatitis B vaccine is immunogenic in patients with diabetes and has a similar safety profile to vaccination in healthy controls. Because increasing age was generally associated with a reduction in seroprotection rates, hepatitis B vaccine should be administered as soon as possible after the diagnosis of diabetes.

Studies on hydromineral balance in fishes frequently employ measurements of electrolytes following euthanasia. We tested the effects of fresh- or salt-water euthanasia baths of tricaine mesylate (MS-222) on plasma magnesium (Mg(2+)) and sodium (Na(+)) ions, cortisol and osmolality in fish exposed to saltwater challenges, and the ion and steroid hormone fluctuations over time following euthanasia in juvenile spring Chinook salmon (Oncorhynchus tshawytscha). Salinity of the euthanasia bath affected plasma Mg(2+) and Na(+) concentrations as well as osmolality, with higher concentrations in fish euthanized in saltwater. Time spent in the bath positively affected plasma Mg(2+) and osmolality, negatively affected cortisol, and had no effect on Na(+) concentrations. The difference of temporal trends in plasma Mg(2+) and Na(+) suggests that Mg(2+) may be more sensitive to physiological changes and responds more rapidly than Na(+). When electrolytes and cortisol are measured as endpoints after euthanasia, care needs to be taken relative to time after death and the salinity of the euthanasia bath., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Adult Chinook salmon (Oncorhynchus tshawytscha) migrate from salt water to freshwater streams to spawn. Immune responses in migrating adult salmon are thought to diminish in the run up to spawning, though the exact mechanisms for diminished immune responses remain unknown. Here we examine both adaptive and innate immune responses as well as pathogen burdens in migrating adult Chinook salmon in the Upper Willamette River basin. Messenger RNA transcripts encoding antibody heavy chain molecules slightly diminish as a function of time, but are still present even after fish have successfully spawned. In contrast, the innate anti-bacterial effector proteins present in fish plasma rapidly decrease as spawning approaches. Fish also were examined for the presence and severity of eight different pathogens in different organs. While pathogen burden tended to increase during the migration, no specific pathogen signature was associated with diminished immune responses. Transcript levels of the immunosuppressive cytokines IL-10 and TGF beta were measured and did not change during the migration. These results suggest that loss of immune functions in adult migrating salmon are not due to pathogen infection or cytokine-mediated immune suppression, but is rather part of the life history of Chinook salmon likely induced by diminished energy reserves or hormonal changes which accompany spawning., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Most pathogen detection tests are imperfect, with a sensitivity < 100%, thereby resulting in the potential for a false negative, where a pathogen is present but not detected. False negatives in a sample inflate the number of non-detections, negatively biasing estimates of pathogen prevalence. Histological examination of tissues as a diagnostic test can be advantageous as multiple pathogens can be examined and providing important information on associated pathological changes to the host. However, it is usually less sensitive than molecular or microbiological tests for specific pathogens. Our study objectives were to 1) develop a hierarchical occupancy model to examine pathogen prevalence in spring Chinook salmon Oncorhynchus tshawytscha and their distribution among host tissues 2) use the model to estimate pathogen-specific test sensitivities and infection rates, and 3) illustrate the effect of using replicate within host sampling on sample sizes required to detect a pathogen. We examined histological sections of replicate tissue samples from spring Chinook salmon O. tshawytscha collected after spawning for common pathogens seen in this population: Apophallus/echinostome metacercariae, Parvicapsula minibicornis, Nanophyetus salmincola/ metacercariae, and Renibacterium salmoninarum. A hierarchical occupancy model was developed to estimate pathogen and tissue-specific test sensitivities and unbiased estimation of host- and organ-level infection rates. Model estimated sensitivities and host- and organ-level infections rates varied among pathogens and model estimated infection rate was higher than prevalence unadjusted for test sensitivity, confirming that prevalence unadjusted for test sensitivity was negatively biased. The modeling approach provided an analytical approach for using hierarchically structured pathogen detection data from lower sensitivity diagnostic tests, such as histology, to obtain unbiased pathogen prevalence estimates with associated uncertainties. Accounting for test sensitivity using within host replicate samples also required fewer individual fish to be sampled. This approach is useful for evaluating pathogen or microbe community dynamics when test sensitivity is <100%.

Assessing the passage of aquatic organisms through culvert road crossings has become increasingly common in efforts to restore stream habitat. Several federal and state agencies and local stakeholders have adopted assessment approaches based on literature-derived criteria for culvert impassability. However, criteria differ and are typically specific to larger-bodied fishes. In an analysis to prioritize culverts for remediation to benefit imperiled, small-bodied fishes in the Upper Coosa River system in the southeastern United States, we assessed the sensitivity of prioritization to the use of differing but plausible criteria for culvert impassability. Using measurements at 256 road crossings, we assessed culvert impassability using four alternative criteria sets represented in Bayesian belief networks. Two criteria sets scored culverts as either passable or impassable based on alternative thresholds of culvert characteristics (outlet elevation, baseflow water velocity). Two additional criteria sets incorporated uncertainty concerning ability of small-bodied fishes to pass through culverts and estimated a probability of culvert impassability. To prioritize culverts for remediation, we combined estimated culvert impassability with culvert position in the stream network relative to other barriers to compute prospective gain in connected stream habitat for the target fish species. Although four culverts ranked highly for remediation regardless of which criteria were used to assess impassability, other culverts differed widely in priority depending on criteria. Our results emphasize the value of explicitly incorporating uncertainty into criteria underlying remediation decisions. Comparing outcomes among alternative, plausible criteria may also help to identify research most needed to narrow management uncertainty.

The southeastern United States has experienced severe, recurrent drought, rapid human population growth, and increasing agricultural irrigation during recent decades, resulting in greater demand for the water resources. During the same time period, freshwater mussels (Unioniformes) in the region have experienced substantial population declines. Consequently, there is growing interest in determining how mussel population declines are related to activities associated with water resource development. Determining the causes of mussel population declines requires, in part, an understanding of the factors influencing mussel population dynamics. We developed Pradel reverse-time, tag-recapture models to estimate survival, recruitment, and population growth rates for three federally endangered mussel species in the Apalachicola-Chattahoochee-Flint River Basin, Georgia. The models were parameterized using mussel tag-recapture data collected over five consecutive years from Sawhatchee Creek, located in southwestern Georgia. Model estimates indicated that mussel survival was strongly and negatively related to high flows during the summer, whereas recruitment was strongly and positively related to flows during the spring and summer. Using these models, we simulated mussel population dynamics under historic (1940-1969) and current (1980-2008) flow regimes and under increasing levels of water use to evaluate the relative effectiveness of alternative minimum flow regulations. The simulations indicated that the probability of simulated mussel population extinction was at least 8 times greater under current hydrologic regimes. In addition, simulations of mussel extinction under varying levels of water use indicated that the relative risk of extinction increased with increased water use across a range of minimum flow regulations. The simulation results also indicated that our estimates of the effects of water use on mussel extinction were influenced by the assumptions about the dynamics of the system, highlighting the need for further study of mussel population dynamics.

Modifications to stream hydrologic regimes can have a profound influence on the dynamics of their fish populations. Using hierarchical linear models, we examined the relations between flow regime and young-of-year fish density using fish sampling and discharge data from three different warmwater streams in Illinois, Alabama, and Georgia. We used an information theoretic approach to evaluate the relative support for models describing hypothesized influences of five flow regime components representing: short-term high and low flows; short-term flow stability; and long-term mean flows and flow stability on fish reproductive success during fish spawning and rearing periods. We also evaluated the influence of ten fish species traits on fish reproductive success. Species traits included spawning duration, reproductive strategy, egg incubation rate, swimming locomotion morphology, general habitat preference, and food habits. Model selection results indicated that young-of-year fish density was positively related to short-term high flows during the spawning period and negatively related to flow variability during the rearing period. However, the effect of the flow regime components varied substantially among species, but was related to species traits. The effect of short-term high flows on the reproductive success was lower for species that broadcast their eggs during spawning. Species with cruiser swimming locomotion morphologies (e.g., Micropterus) also were more vulnerable to variable flows during the rearing period. Our models provide insight into the conditions and timing of flows that influence the reproductive success of warmwater stream fishes and may guide decisions related to stream regulation and management.

In the present study, specimens of the invasive clam, Corbicula fluminea, were collected above and below possible sources of potentially toxic trace elements (As, Cd, Cr, Cu, Hg, Pb, and Zn) in the Altamaha River system (Georgia, U.S.A.). Bioaccumulation of these elements was quantified, along with environmental (water and sediment) concentrations. Hierarchical linear models were used to account for variability in tissue concentrations related to environmental (site water chemistry and sediment characteristics) and individual (growth metrics) variables while identifying the strongest relations between these variables and trace element accumulation. The present study found significantly elevated concentrations of Cd, Cu, and Hg downstream of the outfall of kaolin-processing facilities, Zn downstream of a tire cording facility, and Cr downstream of both a nuclear power plant and a paper pulp mill. Models of the present study indicated that variation in trace element accumulation was linked to distance upstream from the estuary, dissolved oxygen, percentage of silt and clay in the sediment, elemental concentrations in sediment, shell length, and bivalve condition index. By explicitly modeling environmental variability, the Hierarchical linear modeling procedure allowed the identification of sites showing increased accumulation of trace elements that may have been caused by human activity. Hierarchical linear modeling is a useful tool for accounting for environmental and individual sources of variation in bioaccumulation studies.

Aiming to improve upon previously disclosed Factor Xa inhibitors, a series of 4,4-disubstituted pyrrolidine-1,2-dicarboxamides were explored with the intent of increasing the projected human half-life versus 5 (projected human t(1/2)=6 h). A stereospecific route to compounds containing a 4-aryl-4-hydroxypyrrolidine scaffold was developed, resulting in several compounds that demonstrated an increase in the half-life as well as an increase in the in vitro potency compared to 5. Reported herein is the discovery of 26, containing a (2R,4S)-4-hydroxy-4-(2,4-difluorophenyl)-pyrrolidine scaffold, which is a selective, orally bioavailable, efficacious Factor Xa inhibitor that appears suitable for a once-daily dosing (projected human t(1/2)=23 h).

Osteopontin (OPN) plays an important role in left ventricular (LV) remodeling after myocardial infarction (MI) by promoting collagen synthesis and accumulation. This study tested the hypothesis that MMP inhibition modulates post-MI LV remodeling in mice lacking OPN. Wild-type (WT) and OPN knockout (KO) mice were treated daily with MMP inhibitor (PD166793, 30 mg/kg/day) starting 3 days post-MI. LV functional and structural remodeling was measured 14 days post-MI. Infarct size was similar in WT and KO groups with or without MMP inhibition. M-mode echocardiography showed greater increase in LV end-diastolic (LVEDD) and end-systolic diameters (LVESD) and decrease in percent fractional shortening (%FS) and ejection fraction in KO-MI versus WT-MI. MMP inhibition decreased LVEDD and LVESD, and increased %FS in both groups. Interestingly, the effect was more pronounced in KO-MI group versus WT-MI (P < 0.01). MMP inhibition significantly decreased post-MI LV dilation in KO-MI group as measured by Langendorff-perfusion analysis. MMP inhibition improved LV developed pressures in both MI groups. However, the improvement was significantly higher in KO-MI group versus WT-MI (P < 0.05). MMP inhibition increased heart weight-to-body weight ratio, myocyte cross-sectional area, fibrosis and septal wall thickness only in KO-MI. Percent apoptotic myocytes in the non-infarct area was not different between the treatment groups. Expression and activity of MMP-2 and MMP-9 in the non-infarct area was higher in KO-MI group 3 days post-MI. MMP inhibition reduced MMP-2 activity in KO-MI with no effect on the expression of TIMP-2 and TIMP-4 14 days post-MI. Thus, activation of MMPs contributes to reduced fibrosis and LV dysfunction in mice lacking OPN.

Perfluoroalkyl acids (PFAAs) are widespread contaminants emanating from, among other sources, the production/degradation of fluorinated chemicals used in surface repellant applications, such as carpet manufacturing. The goal of the present study was to assess the concentrations of PFAAs, including perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUA), and perfluorooctane sulfonamide (PFOSA), in surface waters both near a wastewater land application system (LAS) in Dalton (GA, USA), home to North America's largest carpet manufacturing site, and distant to this location (Altamaha River, GA, USA) to understand the fate of PFAAs in freshwater. Levels of PFAAs were high in the Conasauga River (GA, USA) downstream of the LAS (PFOA, 253-1,150 ng/L; PFOS, 192-318 ng/L; PFNA, 202-369 ng/L; PFDA, 30.1-113 ng/L; PFUA, 58.0-99.2 ng/L; PFOSA, 162-283 ng/L) and in streams and ponds in Dalton (PFOA, 49.9-299 ng/L; PFOS, 15.8-120 ng/L), and were among the highest measured at a nonspill or direct-release location. Perfluoroalkyl acids in the Altamaha River were much lower (PFOA, 3.0-3.1 ng/L; PFOS, 2.6-2.7 ng/L), but were a source of PFAAs to Georgia's estuaries. A preliminary hazard assessment indicated that concentrations of PFOS at two sites in the Conasauga River exceeded the threshold effect predicted for birds consuming aquatic organisms that are exposed continuously to the PFOS levels at these sites. Assuming that toxicity for all PFAAs quantified is equal to that of PFOS, the sum total PFAAs at two sites within the Conasauga River exceeded PFOS thresholds for aquatic and avian species, warranting additional research.

Increased salinity in spawning and nursery grounds in the Savannah River estuary was cited as the primary cause of a 97% decrease in adult striped bass (Morone saxatilis) and a concomitant 96% decrease in striped bass egg production. Restoration efforts focused on environmental remediation and stock enhancement have resulted in restored salinity patterns and increased egg and adult abundances. However, future water needs or harbor development may preclude further recovery by reducing freshwater inflow or increasing salinity intrusion. To assess the effect of potential changes in the salinity regime, we developed models relating discharge, tidal phase, and salinity to striped bass egg and early larval survival and re-cast these in a quantitative Bayesian belief network. The model indicated that a small upstream shift (< or =1.67 km) in the salinity regime would have the least impact on striped bass early life history survival, whereas shifts >1.67 km would have progressively larger impacts, with a 8.33-km shift potentially reducing our estimated survival probability by >28%. Such an impact could have cumulative and long-term detrimental effects on the recovery of the Savannah River striped bass population. The available salinity data were collected during average and low flows, so our model represents some typical and some extreme conditions during a striped bass spawning season. Our model is a relatively simplistic, "first-order" attempt at evaluating potential effects of changes in the Savannah River estuarine salinity regime and points to areas of concern and potential future research.

Herein, we report the discovery of novel, proline-based factor Xa inhibitors containing a neutral P1 chlorophenyl pharmacophore. Through the additional incorporation of 1-(4-amino-3-fluoro-phenyl)-1H-pyridin-2-one 22, as a P4 pharmacophore, we discovered compound 7 (PD 0348292). This compound is a selective, orally bioavailable, efficacious FXa inhibitor that is currently in phase II clinical trials for the treatment and prevention of thrombotic disorders.

Herein, we report on the identification of three potent glycine and related amino acid-based series of FXa inhibitors containing a neutral P1 chlorophenyl pharmacophore. A X-ray crystal structure has shown that constrained glycine derivatives with optimized N-substitution can greatly increase hydrophobic interactions in the FXa active site. Also, the substitution of a pyridone ring for a phenylsulfone ring in the P4 sidechain resulted in an inhibitor with enhanced oral bioavailability.

At least 56 matrix metalloproteinase (MMP) inhibitors have been pursued as clinical candidates since the late 1970's when the first drug discovery program targeting this enzyme family began. Some of these clinical candidates were pursued for multiple indications. However, the two primary indications that have been targeted are cancer (24 drugs) and anti-arthritis (27 drugs). Cardiovascular disease was listed as an indication for 10 drugs. Forty-six MMP inhibitors have been discontinued, 7 remain in clinical development, and only 1 (Periostat for periodontal disease) has been approved. Recently, negative phase II results were reported for the MMP inhibitor PG-116800, which was being evaluated as a treatment for post-ischemic myocardial remodeling to prevent heart failure. One major factor leading to the failure of PG-116800 and many of the other MMP inhibitors is the inadequate assessment of the therapeutic index, the ratio of dose required for efficacy vs. that for toxicology. This review describes the dose-limiting side effect that has hampered MMP inhibitor development (the musculoskeletal syndrome), cardiovascular clinical MMP inhibitor studies, a model of the therapeutic index using marimastat, and progress towards more selective MMP inhibitors not limited by the musculoskeletal syndrome.

Gas6 (encoded by growth arrest-specific gene 6) is a vitamin-K dependent protein highly homologous to coagulation protein S that is secreted from platelet alpha-granules and has recently been demonstrated to participate in platelet thrombus formation. The current study evaluated the contribution of each of the three known Gas6 receptors (Axl, Sky and Mer) in human and mouse platelet function. Flow cytometry analyses confirmed that all three receptors are present on both human and mouse platelets. Pre-incubation of human platelets with either an anti-Gas6 antibody or blocking antibodies to Sky or Mer inhibited platelet aggregation and degranulation responses to both ADP and the PAR-1 activating peptide, SFLLRN, by more than 80%. In contrast, a stimulatory anti-Axl antibody increased activation responses to these agonists, suggesting a potentiating role for Gas6 in platelet activation. Moreover, in a mouse model of thrombosis, administration of Gas6 or Sky blocking antibodies resulted in a decrease in thrombus weight similar to clopidogrel but, unlike clopidogrel, produced no increase in template bleeding. Thus, Gas6 enhances platelet degranulation and aggregation responses through its known receptors, promoting platelet activation and mediating thrombus formation such that its inhibition prevents thrombosis without increasing bleeding.

Glycoprotein Ib-IX-V (GPIb-IX-V) mediates platelet tethering to von Willebrand factor (VWF), recruiting platelets into the thrombus, and activates integrin alphaIIbbeta3 through a pathway that is dependent on Src kinases. In addition, recent reports indicate that activation of alphaIIbbeta3 by VWF is dependent on protein kinase G (PKG) and mitogen-activated protein (MAP) kinases. The present study compares the importance of these signaling pathways in the activation of alphaIIbbeta3 by GPIb-IX-V. In contrast to a recent report, VWF did not promote an increase in cyclic guanosine monophosphate (cGMP), while agents that elevate cGMP, such as the nitrous oxide (NO) donor glyco-SNAP-1 (N-(beta-D-glucopyranosyl)-N2-acetyl-S-nitroso-D,L-penicillaminamide) or the type 5 phosphosdiesterase inhibitor, sildenafil, inhibited rather than promoted activation of alphaIIbbeta3 by GPIb-IX-V and blocked aggregate formation on collagen at an intermediate rate of shear (800 s(-1)). Additionally, sildenafil increased blood flow in a rabbit model of thrombus formation in vivo. A novel inhibitor of the MAP kinase pathway, which is active in plasma, PD184161, had no effect on aggregate formation on collagen under flow conditions, whereas a novel inhibitor of Src kinases, which is also active in plasma, PD173952, blocked this response. These results demonstrate a critical role for Src kinases but not MAP kinases in VWF-dependent platelet activation and demonstrate an inhibitory role for cGMP-elevating agents in regulating this process.

Collagen turnover is a slow process on a biologic timescale with a t$\\frac12$ of 20-27 days that is mediated primarily by the matrix metalloproteinases (MMPs). Low collagen metabolism is not due to an intrinsically low Km of MMPs, but rather due to a highly regulated system of activity. Despite the stability of collagen and MMPs, the articles in this special addition illustrate the importance of this enzyme family in the disease process leading to congestive heart failure. Like MMPs, drug development is a tightly regulated process, and the successful turnover of MMP inhibitors into a marketed drug has also been a slow process on a pharmaceutical timescale. Since the discovery of the archetypal MMP (type 1 collagenase) over four decades ago by Gross and Lapierre, most major pharmaceutical companies have had MMP inhibitor programs for a variety of indications. Despite decades of research, tens of thousands of compounds synthesized and screened, and billions of dollars spent in clinical studies-Periostat (doxycycline hyclate, CollaGenex Pharmaceuticals Inc.) is the only collagenase inhibitor to be successfully launched. In addition, Periostat's approval is currently limited to periodontal disease. This article focuses on some of the lessons to be learned from the failure of so many MMP inhibitors across so many indications, and what potential exists for MMP inhibitors as a drug class, especially for heart failure.

Background: Left ventricular (LV) hypertrophy and dilatation are important compensatory responses to chronic volume overload. Although LV function is initially preserved by these responses, the continued structural remodeling of the myocardium ultimately becomes maladaptive, leading to the development of heart failure. We have shown previously that increased myocardial matrix metalloproteinase (MMP) activity precedes LV dilatation induced by a chronic volume overload. Accordingly, this study focused on the effects of MMP inhibition therapy (PD 166793, 1 mg x kg(-1) x d(-1)) on LV size and function in a rat model of volume overload-induced heart failure., Methods and Results: Rats were divided into the following groups: treated and untreated infrarenal abdominal aortocaval fistula and treated and untreated sham-operated (control). LV weights of both fistula groups were increased above that of the control group (868+/-79 mg; P< or =0.001); LV weights in the treated fistula group, however, were lower than in the untreated fistula group at 8 weeks (1447+/-186 versus 1715+/-279 mg, respectively; P< or =0.012). The marked ventricular dilatation seen in the untreated fistula group was significantly diminished in the treated fistula group, although the increase in LV compliance was similar in both treated and untreated fistula hearts., Conclusions: MMP inhibition significantly attenuates the myocardial remodeling associated with chronic volume overload, as evidenced by prevention of dilatation, a marked reduction in LV hypertrophy, and preservation of ventricular function.

Background: Collagen degradation is the major mechanism of atherosclerotic plaque destabilization. It is unknown whether collagen breakdown is involved into formation of early atherosclerotic lesions., Methods: Current paper describes a novel collagen degradation assay based on a combination of molecular sieving and mass spectroscopy. The first step of the assay consists of the extraction of total collagen from tissue. This extract includes both intact collagen and its breakdown products. Molecular sieving is used to isolate low molecular weight collagen fragments. Since the low molecular weight fraction of the extract may contain some non-collagenous molecular species, the collagen-specific amino acid hydroxyproline is quantified using mass spectroscopy., Results: This assay was validated in various experimental systems with known/predictable level of collagen breakdown in vitro, ex vivo and in vivo. When applied to cholesterol-fed rabbit aorta, it revealed enhanced collagen degradation in rabbit atheromas compared to unaffected aortic regions., Conclusion: A novel assay has been developed to demonstrate enhanced collagen degradation in rabbit atherosclerotic plaques. Accurate quantification of collagen breakdown products should provide a new relevant end point in the analysis of plaque development and stability.

Background: Matrix metalloproteinase (MMP) activation contributes to tissue remodeling in several disease states, and increased MMP activity has been observed in left ventricular (LV) failure. The present study tested the hypothesis that MMP inhibition would influence LV remodeling and function in developing LV failure., Methods and Results: LV size and function were measured in 5 groups of rats: (1) obese male spontaneously hypertensive heart failure rats (SHHF) at 9 months (n=10), (2) SHHF at 13 months (n=12), (3) SHHF rats treated with an MMP inhibitor during months 9 to 13 (PD166793 5 mg. kg(-1). d(-1) PO; n=14), (4) normotensive Wistar-Furth rats (WF) at 9 months (n=12), and (5) WF at 13 months (n=12). Plasma concentrations of the MMP inhibitor (116+/-11 micromol/L) reduced in vitro LV myocardial MMP-2 activity by approximately 100%. LV function and geometry were similar in WF rats at 9 and 13 months. LV peak +dP/dt was unchanged at 9 months in SHHF but by 13 months was reduced in the SHHF group compared with WF (3578+/-477 versus 5983+/-109 mm Hg/s, P

Objective: Characterize the timecourse of matrix metalloproteinase (MMP-1, -2, -3, -7, -9, -11, -12, -13, and -14) and endogenous tissue inhibitors of MMPs (TIMP-1, -2, -3, and -4) upregulation during left ventricular (LV) remodeling following myocardial infarction (MI) in rats., Methods: The descending left coronary artery of male rats (Rattus norvegicus) was ligated to produce a MI. LV function and dilation were assessed from 1 day to 16 weeks post-MI. Protein and mRNA extraction was done on LV samples containing scar and myocardium together. Gelatinase activity was measured by zymography. Westerns were run on the MMPs known to cleave fibrillar collagen in the rat (MMP-8, -13, and -14) as well as TIMP-1, -2, and -4., Results: Average infarct size was 38.6+/-1.1%, and produced LV dysfunction and progressive LV dilation. Thoracic ascites, a marker of congestive heart failure (HF), was not present until 12 weeks post-MI. Upregulation of MMP-2, -8, -9, -13, and -14 and TIMP-1 and TIMP-2 was detected at different timepoints during HF progression. Increased MMP protein levels occurred sometimes without a corresponding elevation in mRNA levels, and increased TIMP mRNA levels without increased protein levels. MMP-13 active form was elevated during the first 2 weeks post-MI while TIMP-1 and TIMP-2 protein levels were not significantly elevated until 2 weeks post-MI. MMP-8 and MMP-14 protein levels increased later during heart failure progression., Conclusion: MMP/TIMP upregulation evolves over time following infarction in the rat LV. Some MMPs were significantly elevated during the first week post-MI (MMP-13, -2, and -9) and another was not until 16 weeks post-MI (MMP-14). The dissociation between LV MMP/TIMP mRNA and protein levels shows that post-translation processing occurs in the rat heart.

Objective: Determine the effect of a matrix metalloproteinase inhibitor (MMPi) and angiotensin converting enzyme inhibitor (ACEi) on collagen, MMP, tissue inhibitors of MMPs (TIMPs) expression in the spontaneously hypertensive heart failure (SHHF) rat., Methods: Six groups were tested: normotensive 9- and 13-month-old Wistar-Furth (WF) rats, 9-month-old SHHFs (compensatory hypertrophy), 13-month-old SHHFs with HF, and 13-month-old SHHFs orally administered with either an MMPi (PD166793, 5 mgkg(-1)day(-1)) or ACEi (quinapril, 10 mgkg(-1)day(-1)) for 4 months. Collagen volume fraction was assessed histomorphometrically. Left ventricular (LV) mRNA [MMP-1,-2,-3,-7,-9,-11,-13,-14; TIMP-1,-2,-3,-4; and collagen alpha1(I) and alpha1(III)] and protein (MMP-2 and MMP-9 zymographic activity; Western blot analysis of MMP-13, and TIMP-1,-2,-4) levels could be quantified., Results: Collagen mRNA levels were elevated in SHHFs compared to age-matched controls, but collagen volume fraction was elevated only in 13-month-old SHHFs (approximately 2x). Only MMP-2 mRNA levels increased significantly with HF. However, MMP-2 and MMP-9 zymographic activity, and MMP-13 protein levels increased. TIMP-1 and TIMP-2 mRNA and protein levels increased, and TIMP-4 protein levels decreased in SHHFs vs. controls. Both drug treatments reduced LV dilation; preserved systolic function; and normalized MMP/TIMP expression. Both drug treatments also reduced collagen volume fraction, but only quinapril reduced collagen mRNA levels and LV hypertrophy., Conclusions: The divergent effect of MMPi and ACEi on collagen mRNA levels and hypertrophy indicate that drug efficacy is mediated by different pathways in the SHHF rat.

The development of congestive heart failure (CHF) is associated with left ventricle (LV) dilation and myocardial remodeling. The matrix metalloproteinases (MMPs) play a significant role in extracellular remodeling, and recent studies have demonstrated increased MMP expression and activity with CHF. Whether increased MMP activity directly contributes to the LV remodeling with CHF remains unknown. Accordingly, this study examined the effects of chronic MMP inhibition (MMPi) on LV size and function during the progression of CHF. Pigs were assigned to the following groups: (1) CHF, rapid pacing for 3 weeks at 240 bpm (n=12); (2) CHF/MMPi, rapid pacing and concomitant MMPi (PD166793, 20 mg/kg per day [n=10]), and (3) control (n=11). With pacing CHF, LV fractional shortening was reduced (19+/-1 versus 45+/-1%), and end-diastolic dimension increased (5.67+/-0.11 versus 3.55+/-0.05 cm), compared with baseline values (P<0.05). In the CHF/MMPi group, LV endocardial shortening increased (25+/-2%) and the end-diastolic dimension was reduced (4.92+/-0.17 cm) compared with CHF-only values (P<0.05). LV midwall shortening was reduced to a comparable degree in the CHF-only and CHF/MMPi groups. LV peak wall stress increased 3-fold with pacing CHF compared with controls and was significantly reduced in the CHF/MMPi group. LV myocardial stiffness was unchanged with CHF but was increased in the CHF/MMPi group. LV myocyte length was increased with pacing CHF compared with controls (180+/-3 versus 125+/-4 microm, P<0.05) and was reduced in the CHF/MMPi group (169+/-4 microm, P<0.05). Basal-state myocyte shortening velocity was reduced with pacing CHF compared with controls (33+/-2 versus 66+/-1 microm/s, P<0.05) and was unchanged in the CHF/MMPi group (31+/-2 microm/s). Using an ex vivo assay system, myocardial MMP activity was increased with pacing CHF and was reduced with chronic MMPi. In summary, concomitant MMPi with developing CHF limited LV dilation and reduced wall stress. These results suggest that increased myocardial MMP activity contributes to LV myocardial remodeling in developing CHF.