Investigating microbial size classes associated with the transmission of stony coral tissue loss disease (SCTLD).

Effective treatment and prevention of any disease necessitates knowledge of the causative agent, yet the causative agents of most coral diseases remain unknown, in part due to the difficulty of distinguishing the pathogenic microbe(s) among the complex microbial backdrop of coral hosts. Stony coral tissue loss disease (SCTLD) is a particularly destructive disease of unknown etiology, capable of transmitting through the water column and killing entire colonies within a matter of weeks. Here we used a previously described method to (i) isolate diseased and apparently healthy coral colonies within individual mesocosms containing filtered seawater with low microbial background levels; (ii) incubate for several days to enrich the water with coral-shed microbes; (iii) use tangential-flow filtration to concentrate the microbial community in the mesocosm water; and then (iv) filter the resulting concentrate through a sequential series of different pore-sized filters. To investigate the size class of microorganism(s) associated with SCTLD transmission, we used 0.8 µm pore size filters to capture microeukaryotes and expelled zooxanthellae, 0.22 µm pore size filters to capture bacteria and large viruses, and 0.025 µm pore size filters to capture smaller viruses. In an attempt to further refine which size fraction(s) contained the transmissible element of SCTLD, we then applied these filters to healthy "receiver" coral fragments and monitored them for the onset of SCTLD signs over three separate experimental runs. However, several factors outside of our control confounded the transmission results, rendering them inconclusive. As the bulk of prior studies of SCTLD in coral tissues have primarily investigated the associated bacterial community, we chose to characterize the prokaryotic community associated with all mesocosm 0.22 µm pore size filters using Illumina sequencing of the V4 region of the 16S rRNA gene. We identified overlaps with prior SCTLD studies, including the presence of numerous previously identified SCTLD bioindicators within our mesocosms. The identification in our mesocosms of specific bacterial amplicon sequence variants that also appear across prior studies spanning different collection years, geographic regions, source material, and coral species, suggests that bacteria may play some role in the disease.

Chemical and genomic characterization of a potential probiotic treatment for stony coral tissue loss disease.

Considered one of the most devastating coral disease outbreaks in history, stony coral tissue loss disease (SCTLD) is currently spreading throughout Florida's coral reefs and the greater Caribbean. SCTLD affects at least two dozen different coral species and has been implicated in extensive losses of coral cover. Here we show Pseudoalteromonas sp. strain McH1-7 has broad-spectrum antibacterial activity against SCTLD-associated bacterial isolates. Chemical analyses indicated McH1-7 produces at least two potential antibacterials, korormicin and tetrabromopyrrole, while genomic analysis identified the genes potentially encoding an L-amino acid oxidase and multiple antibacterial metalloproteases (pseudoalterins). During laboratory trials, McH1-7 arrested or slowed disease progression on 68.2% of diseased Montastraea cavernosa fragments treated (n = 22), and it prevented disease transmission by 100% (n = 12). McH1-7 is the most chemically characterized coral probiotic that is an effective prophylactic and direct treatment for the destructive SCTLD as well as a potential alternative to antibiotic use.

Clinical development and evaluation of a VEGF-D assay in plasma from patients with metastatic colorectal cancer in the RAISE study.

BACKGROUND: Vascular endothelial growth factor (VEGF)-D was identified as a potential predictive biomarker for ramucirumab efficacy in second-line metastatic colorectal cancer using a research use only (RUO) assay. We describe results with a new assay for detecting VEGF-D in human plasma. METHODS: In RAISE (Clinical Trial Registration: NCT01183780), 1072 patients were randomized 1:1 to ramucirumab or placebo plus FOLFIRI. All patients were then randomized 1:2 to marker exploratory (ME) and marker confirmatory (MC) groups, and those with plasma samples were analyzed accordingly. A new assay validated for investigational use only (IUO) was used to measure VEGF-D levels in plasma, which were analyzed for correlation with overall and progression-free survival (OS/PFS). IUO assay data were compared with historical RUO assay data. RESULTS: ME subset analyses determined the optimal cutpoint of 5.4 ng/mL for defining high/low VEGF-D subgroups. In the combined ME/MC placebo arms, OS/PFS were numerically greater for patients with low vs high VEGF-D (OS: 12.8 vs 11.1 months; PFS: 5.6 vs 4.2 months). In patients with high VEGF-D, ramucirumab vs placebo demonstrated a numerically greater improvement in OS and PFS. Differential efficacy by VEGF-D level was statistically significant for PFS, but not OS. CONCLUSION: In patients with high VEGF-D, ramucirumab demonstrated a greater improvement in OS and PFS vs placebo; however, baseline VEGF-D level was not predictive of ramucirumab OS benefit using VEGF-D assay for IUO. The RAISE intent-to-treat results remain valid.

Disease Diagnostics and Potential Coinfections by Vibrio coralliilyticus During an Ongoing Coral Disease Outbreak in Florida.

A deadly coral disease outbreak has been devastating the Florida Reef Tract since 2014. This disease, stony coral tissue loss disease (SCTLD), affects at least 22 coral species causing the progressive destruction of tissue. The etiological agents responsible for SCTLD are unidentified, but pathogenic bacteria are suspected. Virulence screens of 400 isolates identified four potentially pathogenic strains of Vibrio spp. subsequently identified as V. coralliilyticus. Strains of this species are known coral pathogens; however, cultures were unable to consistently elicit tissue loss, suggesting an opportunistic role. Using an improved immunoassay, the VcpA RapidTest, a toxic zinc-metalloprotease produced by V. coralliilyticus was detected on 22.3% of diseased Montastraea cavernosa (n = 67) and 23.5% of diseased Orbicella faveolata (n = 24). VcpA+ corals had significantly higher mortality rates and faster disease progression. For VcpA- fragments, 21.6% and 33.3% of M. cavernosa and O. faveolata, respectively, died within 21 d of observation, while 100% of similarly sized VcpA+ fragments of both species died during the same period. Further physiological and genomic analysis found no apparent differences between the Atlantic V. coralliilyticus strains cultured here and pathogens from the Indo-Pacific but highlighted the diversity among strains and their immense genetic potential. In all, V. coralliilyticus may be causing coinfections that exacerbate existing SCTLD lesions, which could contribute to the intraspecific differences observed between colonies. This study describes potential coinfections contributing to SCTLD virulence as well as diagnostic tools capable of tracking the pathogen involved, which are important contributions to the management and understanding of SCTLD.

Provisional standardization of hepcidin assays: creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference material.

Background Hepcidin concentrations measured by various methods differ considerably, complicating interpretation. Here, a previously identified plasma-based candidate secondary reference material (csRM) was modified into a serum-based two-leveled sRM. We validated its functionality to increase the equivalence between methods for international standardization. Methods We applied technical procedures developed by the International Consortium for Harmonization of Clinical Laboratory Results. The sRM, consisting of lyophilized serum with cryolyoprotectant, appeared commutable among nine different measurement procedures using 16 native human serum samples in a first round robin (RR1). Harmonization potential of the sRM was simulated in RR1 and evaluated in practice in RR2 among 11 measurement procedures using three native human plasma samples. Comprehensive purity analysis of a candidate primary RM (cpRM) was performed by state of the art procedures. The sRM was value assigned with an isotope dilution mass spectrometry-based candidate reference method calibrated using the certified pRM. Results The inter-assay CV without harmonization was 42.1% and 52.8% in RR1 and RR2, respectively. In RR1, simulation of harmonization with sRM resulted in an inter-assay CV of 11.0%, whereas in RR2 calibration with the material resulted in an inter-assay CV of 19.1%. Both the sRM and pRM passed international homogeneity criteria and showed long-term stability. We assigned values to the low (0.95±0.11 nmol/L) and middle concentration (3.75±0.17 nmol/L) calibrators of the sRM. Conclusions Standardization of hepcidin is possible with our sRM, which value is assigned by a pRM. We propose the implementation of this material as an international calibrator for hepcidin.

Analytical Characterization of an Enzyme-Linked Immunosorbent Assay for the Measurement of Transforming Growth Factor ß1 in Human Plasma.

BACKGROUND: The transforming growth factor ß (TGF-ß)-signaling pathway has emerged as a promising therapeutic target for many disease states including hepatocellular carcinoma (HCC). Because of the pleiotropic effects of this pathway, patient selection and monitoring may be important. TGF-ß1 is the most prevalent isoform, and an assay to measure plasma levels of TGF-ß1 would provide a rational biomarker to assist with patient selection. Therefore, the objective of this study was to analytically validate a colorimetric ELISA for the quantification of TGF-ß1 in human plasma. METHODS: A colorimetric sandwich ELISA for TGF-ß1 was analytically validated per Clinical and Laboratory Standards Institute protocols by assessment of precision, linearity, interfering substances, and stability. A reference range for plasma TGF-ß1 was established for apparently healthy individuals and potential applicability was demonstrated in HCC patients. RESULTS: Precision was assessed for samples ranging from 633 to 10822 pg/mL, with total variance ranging from 28.4% to 7.2%. The assay was linear across the entire measuring range, and no interference of common blood components or similar molecules was observed. For apparently healthy individuals, the average TGF-ß1 level was 1985 ± 1488 pg/mL compared to 4243 ± 2003 pg/mL for HCC patients. Additionally, the TGF-ß1 level in plasma samples was demonstrated to be stable across all conditions tested, including multiple freeze-thaw cycles. CONCLUSIONS: The ELISA described in this report is suitable for the quantification of TGF-ß1 in human plasma and for investigational use in an approved clinical study.

Analytical Validation of the ReEBOV Antigen Rapid Test for Point-of-Care Diagnosis of Ebola Virus Infection.

BACKGROUND: Ebola virus disease (EVD) is a severe viral illness caused by Ebola virus (EBOV). The 2013-2016 EVD outbreak in West Africa is the largest recorded, with >11 000 deaths. Development of the ReEBOV Antigen Rapid Test (ReEBOV RDT) was expedited to provide a point-of-care test for suspected EVD cases. METHODS: Recombinant EBOV viral protein 40 antigen was used to derive polyclonal antibodies for RDT and enzyme-linked immunosorbent assay development. ReEBOV RDT limits of detection (LOD), specificity, and interference were analytically validated on the basis of Food and Drug Administration (FDA) guidance. RESULTS: The ReEBOV RDT specificity estimate was 95% for donor serum panels and 97% for donor whole-blood specimens. The RDT demonstrated sensitivity to 3 species of Ebolavirus (Zaire ebolavirus, Sudan ebolavirus, and Bundibugyo ebolavirus) associated with human disease, with no cross-reactivity by pathogens associated with non-EBOV febrile illness, including malaria parasites. Interference testing exhibited no reactivity by medications in common use. The LOD for antigen was 4.7 ng/test in serum and 9.4 ng/test in whole blood. Quantitative reverse transcription-polymerase chain reaction testing of nonhuman primate samples determined the range to be equivalent to 3.0 × 105-9.0 × 108 genomes/mL. CONCLUSIONS: The analytical validation presented here contributed to the ReEBOV RDT being the first antigen-based assay to receive FDA and World Health Organization emergency use authorization for this EVD outbreak, in February 2015.

Field Validation of the ReEBOV Antigen Rapid Test for Point-of-Care Diagnosis of Ebola Virus Infection.

BACKGROUND: The 2013-2016 West African Ebola virus disease (EVD) epidemic is the largest recorded. Triage on the basis of clinical signs had limited success, and the time to diagnosis by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) could exceed 5 days. Here we describe the development and field validation of the ReEBOV Antigen Rapid Test (ReEBOV RDT) to aid triage of individuals with suspected EVD. METHODS: Samples from patients with suspected EVD were submitted to Kenema Government Hospital, Sierra Leone, for Lassa fever and EVD screening throughout 2014. Banked residual clinical samples were tested in November 2014 and January 2015 in a blinded field trial to estimate the clinical effectiveness of the ReEBOV RDT, compared with EBOV-specific qRT-PCR. RESULTS: Preliminary ReEBOV RDT performance demonstrated a positive percentage agreement (PPA) of 91.1% (195 of 214 results; 95% confidence interval [CI], 86.5%-94.6%) and a negative percentage agreement (NPA) of 90.2% (175 of 194; 95% CI, 85.1%-94.0%). The final estimates used by the Food and Drug Administration to determine whether to grant emergency use authorization for the test, which excluded a qRT-PCR reference method threshold cutoff, were a PPA of 62.1% (72 of 116 results; 95% CI, 52.6%-70.9%) and a NPA of 96.7% (58 of 60; 95% CI, 88.5%-99.6%), with a diagnostic likelihood of 18.6. A subsequent, independent evaluation by the World Health Organization generated results consistent with the preliminary performance estimates. CONCLUSIONS: The ReEBOV RDT demonstrated the potential to provide clinically effective rapid and accurate point-of-care test results and, thus, to be a powerful tool for increasing triage efficiency.

Toward Worldwide Hepcidin Assay Harmonization: Identification of a Commutable Secondary Reference Material.

BACKGROUND: Absolute plasma hepcidin concentrations measured by various procedures differ substantially, complicating interpretation of results and rendering reference intervals method dependent. We investigated the degree of equivalence achievable by harmonization and the identification of a commutable secondary reference material to accomplish this goal. METHODS: We applied technical procedures to achieve harmonization developed by the Consortium for Harmonization of Clinical Laboratory Results. Eleven plasma hepcidin measurement procedures (5 mass spectrometry based and 6 immunochemical based) quantified native individual plasma samples (n = 32) and native plasma pools (n = 8) to assess analytical performance and current and achievable equivalence. In addition, 8 types of candidate reference materials (3 concentrations each, n = 24) were assessed for their suitability, most notably in terms of commutability, to serve as secondary reference material. RESULTS: Absolute hepcidin values and reproducibility (intrameasurement procedure CVs 2.9%-8.7%) differed substantially between measurement procedures, but all were linear and correlated well. The current equivalence (intermeasurement procedure CV 28.6%) between the methods was mainly attributable to differences in calibration and could thus be improved by harmonization with a common calibrator. Linear regression analysis and standardized residuals showed that a candidate reference material consisting of native lyophilized plasma with cryolyoprotectant was commutable for all measurement procedures. Mathematically simulated harmonization with this calibrator resulted in a maximum achievable equivalence of 7.7%. CONCLUSIONS: The secondary reference material identified in this study has the potential to substantially improve equivalence between hepcidin measurement procedures and contributes to the establishment of a traceability chain that will ultimately allow standardization of hepcidin measurement results.

Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits.

Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design.

Outcomes of patients with severe sepsis after the first 6 hours of resuscitation at a regional referral hospital in Uganda.

INTRODUCTION: The optimal resuscitation strategy for patients with severe sepsis in resource-limited settings is unknown. Therefore, we determined the association between intravenous fluids, changes in vital signs and lactate after the first 6 hours of resuscitation from severe sepsis, and in-hospital mortality at a hospital in Uganda. MATERIALS AND METHODS: We enrolled patients admitted with severe sepsis to Mbarara Regional Referral Hospital and obtained vital signs and point-of-care blood lactate concentration at admission and after 6 hours of resuscitation. We used logistic regression to determine predictors of in-hospital mortality. RESULTS: We enrolled 218 patients and had 6 hour postresuscitation data for 202 patients. The median (interquartile range) age was 35 (26-50) years, 49% of patients were female, and 57% were HIV infected. The in-hospital mortality was 32% and was associated with admission Glasgow Coma Score (adjusted odds ratio [aOR], 0.749; 95% confidence interval [CI], 0.642-0.875; P < .001), mid-upper arm circumference (aOR, 0.876; 95% CI, 0.797-0.964; P = .007), and 6-hour systolic blood pressure (aOR, 0.979; 95% CI, 0.963-0.995; P = .009) but not lactate clearance of 10% or greater (aOR, 1.2; 95% CI, 0.46-3.10; P = .73). CONCLUSIONS: In patients with severe sepsis in Uganda, obtundation and wasting were more closely associated with in-hospital mortality than lactate clearance of 10% or greater.

Development of Prototype Filovirus Recombinant Antigen Immunoassays.

BACKGROUND: Throughout the 2014-2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples. METHODS: Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development. RESULTS: Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus-specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone. CONCLUSIONS: The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins.

Multiple circulating infections can mimic the early stages of viral hemorrhagic fevers and possible human exposure to filoviruses in Sierra Leone prior to the 2014 outbreak.

Lassa fever (LF) is a severe viral hemorrhagic fever caused by Lassa virus (LASV). The LF program at the Kenema Government Hospital (KGH) in Eastern Sierra Leone currently provides diagnostic services and clinical care for more than 500 suspected LF cases per year. Nearly two-thirds of suspected LF patients presenting to the LF Ward test negative for either LASV antigen or anti-LASV immunoglobulin M (IgM), and therefore are considered to have a non-Lassa febrile illness (NLFI). The NLFI patients in this study were generally severely ill, which accounts for their high case fatality rate of 36%. The current studies were aimed at determining possible causes of severe febrile illnesses in non-LF cases presenting to the KGH, including possible involvement of filoviruses. A seroprevalence survey employing commercial enzyme-linked immunosorbent assay tests revealed significant IgM and IgG reactivity against dengue virus, chikungunya virus, West Nile virus (WNV), Leptospira, and typhus. A polymerase chain reaction-based survey using sera from subjects with acute LF, evidence of prior LASV exposure, or NLFI revealed widespread infection with Plasmodium falciparum malaria in febrile patients. WNV RNA was detected in a subset of patients, and a 419 nt amplicon specific to filoviral L segment RNA was detected at low levels in a single patient. However, 22% of the patients presenting at the KGH between 2011 and 2014 who were included in this survey registered anti-Ebola virus (EBOV) IgG or IgM, suggesting prior exposure to this agent. The 2014 Ebola virus disease (EVD) outbreak is already the deadliest and most widely dispersed outbreak of its kind on record. Serological evidence reported here for possible human exposure to filoviruses in Sierra Leone prior to the current EVD outbreak supports genetic analysis that EBOV may have been present in West Africa for some time prior to the 2014 outbreak.

Differential assay reactivity of immunglobulin A anti-ß2 glycoprotein I antibodies: implications for the clinical interpretation of antiphospholipid antibody testing.

OBJECTIVE: The routine measurement of IgA anticardiolipin (aCL) and IgA anti-ß2 glycoprotein I (anti-ß2 GPI) antibodies remain controversial despite several studies demonstrating an association with thromboembolic disease in patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). This controversy may be a contributing factor for the current under use of IgA antiphospholipid antibodies. We aimed to investigate the nature of discrepant IgA anti-ß2 GPI reactivity to help define the diagnostic value of IgA antiphospholipid antibodies. MATERIAL AND METHODS: Four sera selected from SLE/APS patients and positive for antiphospholipid antibodies but having discrepant IgA anti-ß2 GPI reactivity on two commercial assays were studied. IgA antibodies were affinity purified to investigate anti-ß2 GPI reactivity. Column wash through and eluent fractions were tested on both IgA anti-ß2 GPI assays. Results were normalized to total protein. Assay conjugates and standards from the discrepant assays were interchanged. RESULTS: The diseased samples were strongly positive in one assay [144-388 IgA antiphospholipid (APL) units] and negative or weakly positive in another assay (9.9-53 APL units). IgA eluents from IgA anti-ß2 GPI positive samples reacted 10 times stronger on the reactive assay. When normalized to protein content, the eluents showed no cross-reactivity for IgG or IgM anti-ß2 GPI antibodies, confirming IgA isotype specificity. Conjugate interchange confirmed that both assays bound IgA anti-ß2 GPI antibodies, but the anti-IgA conjugate from the reactive assay was 4 times stronger, suggesting that its ability to detect IgA anti-ß2 GPI antibodies was partially dependent on the anti-IgA conjugate and calibration. CONCLUSION: These results confirm not only the presence of IgA anti-ß2 GPI antibodies in the selected patient samples but also highlight an IgA conjugate issue for the unreactive assay, causing an underestimation of IgA anti-ß2 GPI. This finding may assist in the ongoing standardization efforts of APS antibody testing. In addition, conclusions from published clinical studies may need to be revised as some assays may understate IgA significance.

Platelet thromboxane (11-dehydro-Thromboxane B2) and aspirin response in patients with diabetes and coronary artery disease.

Aspirin (ASA) irreversibly inhibits platelet cyclooxygenase-1 (COX-1) leading to decreased thromboxane-mediated platelet activation. The effect of ASA ingestion on thromboxane generation was evaluated in patients with diabetes (DM) and cardiovascular disease. Thromboxane inhibition was assessed by measuring the urinary excretion of 11-dehydro-thromboxane B2 (11dhTxB2), a stable metabolite of thromboxane A2. The mean baseline urinary 11dhTxB2 of DM was 69.6% higher than healthy controls (P = 0.024): female subjects (DM and controls) had 50.9% higher baseline 11dhTxB2 than males (P = 0.0004), while age or disease duration had no influence. Daily ASA ingestion inhibited urinary 11dhTxB2 in both DM (71.7%) and controls (75.1%, P < 0.0001). Using a pre-established cut-off of 1500 pg/mg of urinary 11dhTxB2, there were twice as many ASA poor responders (ASA "resistant") in DM than in controls (14.8% and 8.4%, respectively). The rate of ASA poor responders in two populations of acute coronary syndrome (ACS) patients was 28.6 and 28.7%, in spite of a significant (81.6%) inhibition of urinary 11dhTxB2 (P < 0.0001). Both baseline 11dhTxB2 levels and rate of poor ASA responders were significantly higher in DM and ACS compared to controls. Underlying systemic oxidative inflammation may maintain platelet function in atherosclerotic cardiovascular disease irrespective of COX-1 pathway inhibition and/or increase systemic generation of thromboxane from non-platelet sources.

Lassa fever in post-conflict sierra leone.

BACKGROUND: Lassa fever (LF), an often-fatal hemorrhagic disease caused by Lassa virus (LASV), is a major public health threat in West Africa. When the violent civil conflict in Sierra Leone (1991 to 2002) ended, an international consortium assisted in restoration of the LF program at Kenema Government Hospital (KGH) in an area with the world's highest incidence of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Clinical and laboratory records of patients presenting to the KGH Lassa Ward in the post-conflict period were organized electronically. Recombinant antigen-based LF immunoassays were used to assess LASV antigenemia and LASV-specific antibodies in patients who met criteria for suspected LF. KGH has been reestablished as a center for LF treatment and research, with over 500 suspected cases now presenting yearly. Higher case fatality rates (CFRs) in LF patients were observed compared to studies conducted prior to the civil conflict. Different criteria for defining LF stages and differences in sensitivity of assays likely account for these differences. The highest incidence of LF in Sierra Leone was observed during the dry season. LF cases were observed in ten of Sierra Leone's thirteen districts, with numerous cases from outside the traditional endemic zone. Deaths in patients presenting with LASV antigenemia were skewed towards individuals less than 29 years of age. Women self-reporting as pregnant were significantly overrepresented among LASV antigenemic patients. The CFR of ribavirin-treated patients presenting early in acute infection was lower than in untreated subjects. CONCLUSIONS/SIGNIFICANCE: Lassa fever remains a major public health threat in Sierra Leone. Outreach activities should expand because LF may be more widespread in Sierra Leone than previously recognized. Enhanced case finding to ensure rapid diagnosis and treatment is imperative to reduce mortality. Even with ribavirin treatment, there was a high rate of fatalities underscoring the need to develop more effective and/or supplemental treatments for LF.

Screening and identification of a novel class of TGF-ß type 1 receptor kinase inhibitor.

Transforming growth factor ß (TGF-ß) type I receptor (activin receptor-like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420,000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration-response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2ß]pyridazin 6-amine) with a low IC(50) value of 0.7 µM. Compound 8 also inhibited the TGF-ß-induced nuclear translocation of SMAD with an EC(50) value of 0.8 µM. Kinetic analysis revealed that compound 8 inhibited ALK5 via mixed-type inhibition, suggesting that it may bind to ALK5 differently than other published adenosine triphosphate site inhibitors.

Darusentan is a potent inhibitor of endothelin signaling and function in both large and small arteries.

Endothelin is a potent vasoconstrictor often up-regulated in hypertension. Endothelin vasoconstriction is mediated via the G-protein coupled endothelin A (ETA) receptor present on vascular smooth muscle. Endothelin receptor antagonists (ERAs) have been shown to antagonize ET-induced vasoconstriction. We describe the primary pharmacology of darusentan, a propanoic acid based ERA currently in phase 3 clinical trials for resistant hypertension. Darusentan was tested in membrane-, cell-, and tissue-based assays to determine its biochemical and functional potency. Rat aortic vascular smooth muscle cells (RAVSMs) were characterized using flow cytometry. RAVSM membrane fractions tested in saturation experiments exhibited moderate endothelin receptor density. Receptor counting revealed that >95% of the endothelin receptors in these fractions were the ETA subtype. (S)-Darusentan competed for radiolabeled endothelin binding in RAVSM membranes with single-site kinetics, exhibiting a Ki = 13 nmol/L. (R)-Darusentan exhibited no binding activity. In cultured RAVSMs, endothelin induced increases in inositol phosphate and Ca2+ signaling, both of which were attenuated by (S)-darusentan in a concentration-dependent manner. In isolated endothelium-denuded rat aortic rings, (S)-darusentan inhibited endothelin-induced vascular contractility with a pA2 = 8.1 +/- 0.14 (n = 4 animals; mean +/- SD). (R)-Darusentan had no effect. The vasorelaxant potency of (S)-darusentan did not change when determined in isolated denuded rat mesenteric arterioles, suggesting a similar mode of action in both conductance and resistance arteries. In vascular smooth muscle, (S)-darusentan is an ERA with high affinity for the ET receptor, which in this preparation is predominantly ETA receptors. (S)-Darusentan inhibits endothelin-induced signaling related to pro-contractile activity and is a potent inhibitor of vasoconstriction in large and small arteries.

Acute effects of endothelin receptor antagonists on hepatic hemodynamics of cirrhotic and noncirrhotic rats.

Hepatic and circulating endothelin-1 (ET-1) are increased in patients with cirrhosis and in cirrhotic animals. However, the distinct roles of ET receptor subtypes ETA and ETB in cirrhosis and portal hypertension (PHT) have not been clearly elucidated. Thus, we studied the effects of selective ET-1 antagonists (ETA-ant or ETB-ant) and nonselective ET-1 antagonist (ETA/B-ant) on hepatic hemodynamics in cirrhotic rats. Liver fibrosis and PHT were induced by complete bile duct ligation (BDL) in rats. Two weeks after BDL or sham surgery, hemodynamic responses were measured during intraportal infusion of incremental doses of the following ET-ants: (i) BQ-123, (ii) BQ-788, and (iii) bosentan. After equilibration with vehicle, doses of ET-ants were infused for 30 min periods, and steady-state systemic and hepatic hemodynamics, portal venous pressure (PVP), and hepatic blood flow (HBF) were measured. BDL induced significant PHT and elevated concentrations of plasma ET-1 compared with sham. ETA-ant decreased PVP of cirrhotic rats but had no effect on sham, whereas ETB-ant increased PVP in sham but had no effect in BDL. Nonselective ETA/B-ant decreased PVP of BDL similarly to ETA-ant. Both ETA-ant and ETB-ant decreased local HBF, whereas a nonselective antagonist did not change HBF in sham; however no significant changes were observed in HBF of BDL rats with any of the antagonists. These findings suggest ETA activation contributes to PHT in cirrhotic rats, whereas ETB-mediated portal depressor effects are attenuated in cirrhotic rats compared with noncirrhotic rats.

Agonist-induced nuclear export of GFP-HDAC5 in isolated adult rat ventricular myocytes.

INTRODUCTION: Histone acetylation/deacetylation represents a central mechanism for the control of gene expression. Histone deacetylases (HDACs) deacetylate histones and transcription factors, causing transcriptional repression critical to the maintenance of the normal adult cardiac myocyte phenotype. Export of HDAC5 from the nucleus is associated with derepression of the fetal gene program and induction of hypertrophy in the cardiac myocyte. Prior studies of HDAC5 localization in adult cardiomyocytes have employed rabbit cells. METHODS: Because many laboratories are restricted from working with USDA-regulated species such as rabbits, we sought to develop a quantitative assay to monitor signal-dependent nuclear export of HDAC5 in adult rat ventricular myocytes (ARVMs). RESULTS: We demonstrate that GFP-tagged HDAC5 expressed from an adenoviral vector appropriately localizes in nuclei of cultured ARVMs within 24 h post infection. Nuclear localized GFP-HDAC5 undergoes quantitative nuclear export (approximately 20-30% within 2 h) in response to hypertrophic agonists such as endothelin-1 (ET-1) and prostaglandin-F2alpha (PGF2alpha), as well as the direct protein kinase C (PKC) activator and phorbol myristate acetate (PMA). Nuclear export of HDAC5 in ARVMs is blocked by Gö6976, a small molecule inhibitor of PKC and protein kinase D (PKD). DISCUSSION: These data suggest that GFP-HDAC5 is appropriately functional in ARVMs 24 h post infection, and that translocation events can be quantitated for the study of hypertrophy or the identification of antihypertrophic agents in adult cardiac myocytes.