Structure and Functional Characterization of a Humanized Anti-CCL20 Antibody following Exposure to Serum Reveals the Formation of Immune Complex That Leads to Toxicity.

mAbs have revolutionized the treatment of autoimmune disorders. Even though mAbs have shown impressive efficacy in blocking T cell or B cell activation and/or recruitment to sites of inflammation, this group of biologicals are not devoid of adverse effects. The most serious adverse effects include infusion reactions, including the activation of the complement pathway. In this study, we present a detailed structure-function study of an anti-CCL20 humanized IgG1 mAb that neutralizes CCL20 chemokine and prevents the recruitment of Th17 cells to sites of inflammation. We demonstrate that the anti-CCL20 Ab changes significantly following administration to humans and monkeys and exposure to human serum. Analysis of the drug product revealed that the anti-CCL20 Ab has unexpectedly high C1q binding. This high binding was linked to immune complex formation in vivo but not during in vitro serum incubation. The immune complex contained multiple complement components. Anti-CCL20 Ab-mediated, complement-dependent cytotoxicity occurred when the Ab bound to CCL20 tethered to the cell membrane of target cells. Taken together, these results provide a likely cause for the animal toxicity observed. In addition, anti-CCL20 revealed progressive acidification because of N100 (located in CDR) deamidation over time, which did not directly impact Ag binding. Our study demonstrates that the safety profiling of mAbs should include the evaluation of effector functions in addition to typical stressed conditions.

Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P.

Lack of simple and robust methods to determine complement activation in human serum induced by antigen-antibody complexes is a major hurdle for monitoring therapeutic antibody drug quality and stability. Dezamizumab is a humanized IgG1 monoclonal antibody that binds to serum amyloid P component (SAP) for potential treatment of systemic amyloidosis. The mechanism of action of Dezamizumab includes the binding of SAP, complement activation through classical pathway, and phagocytosis; however, the steps in this process cannot be easily monitored. We developed two novel methods to determine Dezamizumab-SAP complex-induced complement activation. Complement component 3 (C3) depletion was detected by homogeneous time-resolved fluorescence (HTRF), and C3a desArg fragment, formed after the cleavage of C3 to yield C3a followed by removal of its C-terminal arginine residue, was determined using Meso Scale Discovery (MSD) technology. We found that the presence of both Dezamizumab and SAP was required for complement activation via both methods. The optimal molar ratio of Dezamizumab:SAP was 6:1 in order to obtain maximal complement activation. The relative potency from both methods showed a good correlation to Dezamizumab-SAP-dependent complement component 1q (C1q) binding activity in Dezamizumab thermal-stressed samples. Both SAP and C1q binding, as determined by surface plasmon resonance and the two complement activation potency methods described here, reflect the mechanism of action of Dezamizumab. We conclude that these methods can be used to monitor Dezamizumab quality for drug release and stability testing, and the novel potency methods reported here can be potentially used to evaluate complement activity induced by other antigen-antibody complexes.

Bluetongue virus serotype 12 enters Australia - a further incursion of novel western lineage genome segments.

Bluetongue virus (BTV) is an arbovirus (genus: Orbivirus) that occurs worldwide. It infects domestic and wild ruminant species and can cause disease in livestock, producing high economic impact. Recently, it gained extra prominence throughout Europe, with disease occurring in regions traditionally free of BTV. BTV enters Australia from Southeast Asia via wind-borne infected Culicoides spp. The first Australian isolation was 1975 (BTV-20) and further serotypes were isolated between 1979-86 (BTV-1, -3, -9, -15, -16, -21, -23). Despite increased, more sensitive, monitoring, no more were detected in over two decades, implying a stable BTV episystem of eastern ancestry. Isolations of BTV-2, -7 and -5 then occurred between 2007-15, with the latter two possessing genome segments with high sequence identity to western isolates. We report on the first isolation and genomic characterization of BTV-12, which revealed that three more novel western topotype gene segments have entered northern Australia.

Initiating Titratable Fixed-Ratio Combinations of Basal Insulin Analogs and Glucagon-Like Peptide-1 Receptor Agonists: What You Need to Know.

IN BRIEF Titratable fixed-ratio combinations (FRCs) of a basal insulin and a glucagon-like peptide-1 (GLP-1) receptor agonist are new therapeutic options for people with type 2 diabetes. Two FRCs-insulin degludec/liraglutide and insulin glargine/lixisenatide-have been approved for use in the United States. The two components in these FRCs target different aspects of diabetes pathophysiology, working in a complementary manner to decrease blood glucose while mitigating the side effects associated with each component (hypoglycemia and weight gain with insulin and gastrointestinal side effects with GLP-1 receptor agonists). This article reviews these products and key considerations for their use.

The implementation of a naloxone rescue program in university students.

OBJECTIVE: Responding to the nationwide opioid overdose epidemic, Washington State University initiated a naloxone safety net project intending to increase awareness of opioid overdose, increase the availability of naloxone, and examine university students' perceptions regarding the usefulness of a novel, large-group audience-training model. SETTING: A Washington State University campus. PRACTICE DESCRIPTION: In September 2014, university students were recruited to attended a large-group audience training event which included opioid overdose prevention, recognition, and first response. All trained participants received an intranasal naloxone reversal kit. PRACTICE INNOVATION: Student pharmacists, who previously received naloxone rescue training and overdose education from the pharmacist lead researcher, acted as trainers. The training consisted of a large-group audience delivery with small-group practice sessions facilitated by the student pharmacists. EVALUATION: Participants who attended the recruitment event completed a pre-training survey to assess knowledge and perceptions about opioid use disorder and overdose. The following week, participants attended the training event. Participants were asked to complete a post-training survey to evaluate the usefulness of the program. RESULTS: Forty-three percent of the participants (65/150) who attended the recruitment event reported knowing someone who used prescription opioids to get "high." Seventy-four participants attended the training, and 92% of them (68/74) completed the post-training survey. The majority of respondents agreed that the training program met their expectations and the skills they learned could be used to intervene in an overdose situation. CONCLUSIONS: Before training, survey responses from recruited participates indicated the need to discuss opioid use disorder among university students is important. Use of a training model involving large-group audiences followed by small-group practice sessions offers an acceptable educational solution regarding opioid overdose and prevention. Our experience suggests using this training model to educate university students to recognize and provide first response is a feasible and acceptable approach.

Redox-induced mobilization of copper, selenium, and zinc in deltaic soils originating from Mississippi (U.S.A.) and Nile (Egypt) River Deltas: A better understanding of biogeochemical processes for safe environmental management.

Studies about the mobilization of potentially toxic elements (PTEs) in deltaic soils can be challenging, provide critical information on assessing the potential risk and fate of these elements and for sustainable management of these soils. The impact of redox potential (EH), pH, iron (Fe), manganese (Mn), sulfate (SO42-), chloride (Cl-), aliphatic dissolved organic carbon (DOC), and aromatic dissolved organic carbon (DAC) on the mobilization of copper (Cu), selenium (Se), and zinc (Zn) was studied in two soils collected from the Nile and Mississippi Rivers deltaic plains focused on increasing our understanding of the fate of these toxic elements. Soils were exposed to a range of redox conditions stepwise from reducing to oxidizing soil conditions using an automated biogeochemical microcosm apparatus. Concentrations of DOC and Fe were high under reducing conditions as compared to oxidizing conditions in both soils. The proportion of DAC in relation to DOC in solution (aromaticity) was high in the Nile Delta soil (NDS) and low in the Mississippi Delta soil (MDS) under oxidizing conditions. Mobilization of Cu was low under reducing conditions in both soils which was likely caused by sulfide precipitation and as a result of reduction of Cu2+ to Cu1+. Mobilization of Se was high under low EH in both soils. Release of Se was positively correlated with DOC, Fe, Mn, and SO42- in the NDS, and with Fe in the MDS. Mobilization of Zn showed negative correlations with EH and pH in the NDS while these correlations were non-significant in the MDS. The release dynamics of dissolved Zn could be governed mainly by the chemistry of Fe and Mn in the NDS and by the chemistry of Mn in the MDS. Our findings suggest that a release of Se and Zn occurs under anaerobic conditions, while aerobic conditions favor the release of Cu in both soils. In conclusion, the release of Cu, Se, and Zn under different reducing and oxidizing conditions in deltaic wetland soils should be taken into account due to increased mobilization and the potential environmental risks associated with food security in utilizing these soils for flooded agricultural and fisheries systems.

Advances in Insulin Therapy: A Review of New Insulin Glargine 300 Units/mL in the Management of Diabetes.

In Brief New insulin glargine 300 units/mL (Gla-300) is a formulation of insulin glargine that has a more constant pharmacokinetic profile with a prolonged duration of action. The EDITION clinical trial program showed that the use of Gla-300 leads to glycemic control comparable to that of insulin glargine 100 units/mL in a wide range of populations of people with diabetes. It is associated with comparable to less nocturnal confirmed or severe hypoglycemia and less weight gain, despite requiring a somewhat higher insulin dose than U-100. The distinct pharmacokinetic/pharmacodynamic and clinical profiles of Gla-300 may benefit a range of people with type 1 or type 2 diabetes.

Impacts of Long-Term Irrigation of Domestic Treated Wastewater on Soil Biogeochemistry and Bacterial Community Structure.

Freshwater scarcity and regulations on wastewater disposal have necessitated the reuse of treated wastewater (TWW) for soil irrigation, which has several environmental and economic benefits. However, TWW irrigation can cause nutrient loading to the receiving environments. We assessed bacterial community structure and associated biogeochemical changes in soil plots irrigated with nitrate-rich TWW (referred to as pivots) for periods ranging from 13 to 30 years. Soil cores (0 to 40 cm) were collected in summer and winter from five irrigated pivots and three adjacently located nonirrigated plots. Total bacterial and denitrifier gene abundances were estimated by quantitative PCR (qPCR), and community structure was assessed by 454 massively parallel tag sequencing (MPTS) of small-subunit (SSU) rRNA genes along with terminal restriction fragment length polymorphism (T-RFLP) analysis of nirK, nirS, and nosZ functional genes responsible for denitrification of the TWW-associated nitrate. Soil physicochemical analyses showed that, regardless of the seasons, pH and moisture contents (MC) were higher in the irrigated (IR) pivots than in the nonirrigated (NIR) plots; organic matter (OM) and microbial biomass carbon (MBC) were higher as a function of season but not of irrigation treatment. MPTS analysis showed that TWW loading resulted in the following: (i) an increase in the relative abundance of Proteobacteria, especially Betaproteobacteria and Gammaproteobacteria; (ii) a decrease in the relative abundance of Actinobacteria; (iii) shifts in the communities of acidobacterial groups, along with a shift in the nirK and nirS denitrifier guilds as shown by T-RFLP analysis. Additionally, bacterial biomass estimated by genus/group-specific real-time qPCR analyses revealed that higher numbers of total bacteria, Acidobacteria, Actinobacteria, Alphaproteobacteria, and the nirS denitrifier guilds were present in the IR pivots than in the NIR plots. Identification of the nirK-containing microbiota as a proxy for the denitrifier community indicated that bacteria belonged to alphaproteobacteria from the Rhizobiaceae family within the agroecosystem studied. Multivariate statistical analyses further confirmed some of the above soil physicochemical and bacterial community structure changes as a function of long-term TWW application within this agroecosystem.

Empagliflozin, an SGLT2 inhibitor for the treatment of type 2 diabetes mellitus: a review of the evidence.

OBJECTIVE: To review available studies of empagliflozin, a sodium glucose co-transporter-2 (SGLT2) inhibitor approved in 2014 by the European Commission and the United States Food and Drug Administration for the treatment of type 2 diabetes mellitus (T2DM). DATA SOURCES: PubMed was searched using the search terms empagliflozin, BI 10773, and BI10773, for entries between January 1, 2000, and December 1, 2014. Reference lists from retrieved articles were searched manually for additional peer-reviewed publications. STUDY SELECTION AND DATA EXTRACTION: All publications reporting clinical trials of empagliflozin were eligible for inclusion. DATA SYNTHESIS: Empagliflozin is a new once-daily oral SGLT2 inhibitor with a mechanism of action that is independent of ß-cell function and the insulin pathway. Data from a comprehensive phase III clinical trial program have demonstrated its efficacy as monotherapy, as add-on to other glucose-lowering agents, and in different patient populations. In these studies, empagliflozin resulted in improvements in blood glucose levels as well as reductions in body weight and blood pressure. Empagliflozin was well tolerated and was not associated with an increased risk of hypoglycemia versus placebo. CONCLUSION: The oral antidiabetes agent, empagliflozin, can be used as monotherapy or alongside other glucose-lowering treatments, including insulin, to treat T2DM.

New Insulin Glargine 300 U/mL for the Treatment of Type 1 and Type 2 Diabetes Mellitus.

OBJECTIVE: To describe the studies evaluating the efficacy and safety of new insulin glargine 300 U/mL (Gla-300) as a basal insulin in the treatment of type 1 (T1DM) and type 2 (T2DM) diabetes mellitus. DATA SOURCES: A literature search of MEDLINE was conducted (January 2008-June 2015) using the terms U300, Gla-300, and insulin glargine 300 units/mL and supplemented with congress abstracts published in 2014 and 2015. STUDY SELECTION AND DATA EXTRACTION: All English language studies assessing the efficacy and/or safety of Gla-300 were evaluated. DATA SYNTHESIS: The efficacy and safety of once-daily Gla-300 has been compared with insulin glargine 100 U/mL (Gla-100) in the EDITION trials, 6 phase-3, multinational, open-label studies in T1DM and T2DM. Across these studies, Gla-300 consistently demonstrated glycemic control comparable to Gla-100; a mean (standard error) change in glycated hemoglobin A1c of -1.02% (0.03) with both Gla-100 (n = 1235) and Gla-300 (n = 1239) was seen in a patient-level meta-analysis. Gla-300 was associated with comparable or reduced nocturnal hypoglycemia compared with Gla-100; the relative risk for nocturnal hypoglycemia with Gla-300 versus Gla-100 was 0.75 (95% CI = 0.68 to 0.83) in a patient-level meta-analysis. There is also some evidence for less weight gain with Gla-300 compared with Gla-100, despite a higher insulin dose. Gla-300 was well tolerated, with the number of adverse events being comparable to that with Gla-100. CONCLUSIONS: These results suggest that Gla-300 may have a place as an alternative, long-acting basal insulin for patients with T1DM or T2DM, with the possibility for improved tolerability.

A Brief History of the Development of Diabetes Medications.

This article provides an overview of the development of insulins, oral agents, and noninsulin injectable agents used in the management of hyperglycemia in patients with diabetes. It also briefly reviews the pharmacological impact and salient side effects of these medications.

Vertical dissolved inorganic nitrogen fluxes in marsh and mudflat areas of the yangtze estuary.

Nitrogen (N) is a dominant macronutrient in many river-dominated coastal systems, and excess concentrations can drive eutrophication, the effects of which can include hypoxia and algal blooms. The Yangtze River in China transports a large amount of dissolved inorganic N. Therefore, it is important to understand the role of the marsh and mudflat areas within the estuary on processing this exogenous N load. In situ dissolved inorganic nitrogen (DIN) fluxes across the sediment-water interface were determined monthly at Chongming Island at two sites (a vegetated marsh and an unvegetated mudflat) and were compared with rates from a previously published laboratory incubation study by our research group. Results from the in situ study showed that NO flux rates comprised the major component of total DIN flux, ranging from 55 to 97%. No significant difference was observed in the N flux rates between the marsh and mudflat sites. Overall, sediment at both sites served as a sink of DIN from surface water with mean flux rates of -178 µmol m h and -165 µmol m h for the marsh and mudflat, respectively. In general, DIN flux rates were not significantly correlated with DIN concentrations and other measured parameters (temperature, dissolved oxygen, salinity, and pH) of surface water. The in situ measured fluxes of NO and NO in this study were not significantly different from those of our previous laboratory incubation ( > 0.05), whereas NH fluxes in situ were significantly lower than those from the laboratory core incubations ( < 0.05). This result suggests that caution should be used when extrapolating rates from laboratory incubation methods to the field because the rates might not be equivalent.

Investigation of biogeochemical functional proxies in headwater streams across a range of channel and catchment alterations.

Historically, headwater streams received limited protection and were subjected to extensive alteration from logging, farming, mining, and development activities. Despite these alterations, headwater streams provide essential ecological functions. This study examines proxy measures of biogeochemical function across a range of catchment alterations by tracking nutrient cycling (i.e., inputs, processing, and stream loading) with leaf litter fall, leaf litter decomposition, and water quality parameters. Nutrient input and processing remained highest in second growth forests (the least altered areas within the region), while recently altered locations transported higher loads of nutrients, sediments, and conductivity. Biogeochemical functional proxies of C and N input and processing significantly, positively correlated with rapid assessment results (Pearson coefficient = 0.67-0.81; P = 0.002-0.016). Additionally, stream loading equations demonstrate that N and P transport, sediment, and specific conductivity negatively correlated with rapid assessment scores (Pearson coefficient = 0.56-0.81; P = 0.002-0.048). The observed increase in stream loading with lower rapid assessment scores indicates that catchment alterations impact stream chemistry and that rapid assessments provide useful proxy measures of function in headwater ecosystems. Significant differences in nutrient processing, stream loading, water quality, and rapid assessment results were also observed between recently altered (e.g., mined) headwater streams and older forested catchments (Mann-Whitney U = 24; P = 0.01-0.024). Findings demonstrate that biogeochemical function is reduced in altered catchments, and rapid assessment scores respond to a combination of alteration type and recovery time. An analysis examining time and economic requirements of proxy measurements highlights the benefits of rapid assessment methods in evaluating biogeochemical functions.

Impact of freshwater river reconnection on porewater salinity and ammonium availability in coastal brackish marsh soils.

Restoring freshwater flows to wetland ecosystems is an increasingly common tool for reversing saltwater intrusion/chronic salinization. Hydrologic restoration projects can deliver large volumes of sediment and fresh water to coastal basins, episodically exposing brackish and salt marsh vegetated soils to low surface water salinities. Yet little is known about the impacts of river reconnection/diversions to porewater salinity of the active root zone (0-30 cm) and salinity dependent soil biogeochemical processes like sorption. Intact soil cores from a brackish marsh site in mid-Barataria Basin, LA were subjected to a simulated river diversion opening to examine how porewater salinity and ammonium (NH4+) availability change with depth and time. Quadruplicate soil cores were inundated with continuously flowing fresh (0 salinity) water for 0, 7, or 28 d then measured for porewater salinity and NH4+ partition coefficient (exchangeable NH4+:porewater NH4+) every 2 cm for the top 10 cm of soil. Porewater salinity decreased in the 0-4 cm interval between 0 and 7 d of the simulated river diversion and increased in the 8-10 cm interval between 7 and 28 d. Overall, depth-averaged porewater salinity of the top 10 cm did not significantly change between 0 and 28 d of the simulated river diversion. Ammonium partition coefficients increased only in the 0-2 cm interval between 0 and 7 d of the simulated river diversion, likely due to freshening-induced NH4+ adsorption. These results indicate that the physicochemical environment of brackish marsh soils is relatively resistant to a single surface water freshening over one month. Models utilized by the state of Louisiana may be overpredicting freshening of the marsh soil porewater in Mid-Barataria Basin in response to the episodic operation of the Mid-Barataria Sediment Diversion. This study demonstrates the importance of measuring diffusive-adsorptive flux of major cations and anions when modeling vertical salt transfer in brackish marsh soils.

An industry perspective approach and control strategy for implementation of ready-to-use cells in bioassays: survey outcome and recommendations.

The BioPhorum Development Group is an industry collaboration enabling the sharing of common practices for the development of biopharmaceuticals. Bioassays are an important part of an analytical control system. Utilization of ready-to-use cells can increase operational flexibility and improve efficiency by providing frozen cell banks uniform stock while removing challenges associated with maintaining cultured cells. The BioPhorum Development Group-Bioassay workstream conducted an intercompany benchmarking survey and group discussions around the use of ready-to-use cells for bioassays. The results of the collaboration provide alignment on nomenclature, production, qualification and implementation of ready-to-use cells to support the assay life cycle.

Vaccination of ferrets with a recombinant G glycoprotein subunit vaccine provides protection against Nipah virus disease for over 12 months.

BACKGROUND: Nipah virus (NiV) is a zoonotic virus belonging to the henipavirus genus in the family Paramyxoviridae. Since NiV was first identified in 1999, outbreaks have continued to occur in humans in Bangladesh and India on an almost annual basis with case fatality rates reported between 40% and 100%. METHODS: Ferrets were vaccinated with 4, 20 or 100 µg HeVsG formulated with the human use approved adjuvant, CpG, in a prime-boost regime. One half of the ferrets were exposed to NiV at 20 days post boost vaccination and the other at 434 days post vaccination. The presence of virus or viral genome was assessed in ferret fluids and tissues using real-time PCR, virus isolation, histopathology, and immunohistochemistry; serology was also carried out. Non-immunised ferrets were also exposed to virus to confirm the pathogenicity of the inoculum. RESULTS: Ferrets exposed to Nipah virus 20 days post vaccination remained clinically healthy. Virus or viral genome was not detected in any tissues or fluids of the vaccinated ferrets; lesions and antigen were not identified on immunohistological examination of tissues; and there was no increase in antibody titre during the observation period, consistent with failure of virus replication. Of the ferrets challenged 434 days post vaccination, all five remained well throughout the study period; viral genome - but not virus - was recovered from nasal secretions of one ferret given 20 µg HeVsG and bronchial lymph nodes of the other. There was no increase in antibody titre during the observation period, consistent with lack of stimulation of a humoral memory response. CONCLUSIONS: We have previously shown that ferrets vaccinated with 4, 20 or 100 µg HeVsG formulated with CpG adjuvant, which is currently in several human clinical trials, were protected from HeV disease. Here we show, under similar conditions of use, that the vaccine also provides protection against NiV-induced disease. Such protection persists for at least 12 months post-vaccination, with data supporting only localised and self-limiting virus replication in 2 of 5 animals. These results augur well for acceptability of the vaccine to industry.