Treatment of experimental adjuvant arthritis with a novel folate receptor-targeted folic acid-aminopterin conjugate

Introduction Folate receptor (FR)-expressing macrophages have been shown to accumulate at sites of inflammation, where they promote development of inflammatory symptoms. To target such a macrophage population, we designed and evaluated the biologic activity of EC0746, a novel folic acid conjugate of the highly potent antifolate, aminopterin. Methods Using a FR-positive subclone of murine macrophage-derived RAW264.7 cells and rat thioglycollate-elicited macrophages, we studied the effect of EC0746 on dihydrofolate reductase activity, cell proliferation, and cellular response towards bacterial lipopolysaccharide as well as IFNγ activation. The EC0746 anti-inflammatory activity, pharmacokinetics, and toxicity were also evaluated in normal rats or in rats with adjuvant-induced arthritis; that is, a FR-positive macrophage model that closely resembles rheumatoid arthritis in humans. Results EC0746 suppresses the proliferation of RAW264.7 cells and prevents the ability of nonproliferating rat macrophages to respond to inflammatory stimuli. In the macrophage-rich rat arthritis model, brief treatment with subcutaneously administered EC0746 is shown to mediate an FR-specific anti-inflammatory response that is more potent than either orally administered methotrexate or subcutaneously delivered etanercept. More importantly, EC0746 therapy is also shown to be ~40-fold less toxic than unmodified aminopterin, with fewer bone marrow and gastrointestinal problems. Conclusions EC0746 is the first high FR-binding dihydrofolate reductase inhibitor that demonstrates FR-specific anti-inflammatory activities both in vitro and in vivo. Our data reveal that a relatively toxic anti-inflammatory drug, such as aminopterin, can be targeted with folic acid to inflammatory macrophages and thereby relieve inflammatory symptoms with greatly reduced toxicity

Drone-based Water Sampling and Characterization of Three Freshwater Harmful Algal Blooms in the United States

Freshwater harmful algal blooms (HABs), caused mostly by toxic cyanobacteria, produce a range of cyanotoxins that threaten the health of humans and domestic animals. Climate conditions and anthropogenic influences such as agricultural run-off can alter the onset and intensity of HABs. Little is known about the distribution and spread of freshwater HABs. Current sampling protocols in some lakes involve teams of researchers that collect samples by hand from a boat and/or from the shoreline. Water samples can be collected from the surface, from discrete-depth collections, and/or from depth-integrated intervals. These collections are often restricted to certain months of the year, and generally are only performed at a limited number of collection sites. In lakes with active HABs, surface samples are generally sufficient for HAB water quality assessments. We used a unique DrOne Water Sampling SystEm (DOWSE) to collect water samples from the surface of three different HABs in Ohio (Grand Lake St Marys, GLSM and Lake Erie) and Virginia (Lake Anna), United States in 2019. The DOWSE consisted of a 3D-printed sampling device tethered to a drone (uncrewed aerial system, or UAS), and was used to collect surface water samples at different distances (10–100 m) from the shore or from an anchored boat. One hundred and eighty water samples (40 at GLSM, 20 at Lake Erie, and 120 at Lake Anna) were collected and analyzed from 18 drone flights. Our methods included testing for cyanotoxins, phycocyanin, and nutrients from surface water samples. Mean concentrations of microcystins (MCs) in drone water samples were 15.00, 1.92, and 0.02 ppb for GLSM, Lake Erie, and Lake Anna, respectively. Lake Anna had low levels of anatoxin in nearly all (111/120) of the drone water samples. Mean concentrations of phycocyanin in drone water samples were 687, 38, and 62 ppb for GLSM, Lake Erie, and Lake Anna, respectively. High levels of total phosphorus were observed in the drone water samples from GLSM (mean of 0.34 mg/L) and Lake Erie (mean of 0.12 mg/L). Lake Anna had the highest variability of total phosphorus with concentrations that ranged from 0.01 mg/L to 0.21 mg/L, with a mean of 0.06 mg/L. Nitrate levels varied greatly across sites, inverse with bloom biomass, ranging from below detection to 3.64 mg/L, with highest mean values in Lake Erie followed by GLSM and Lake Anna, respectively. Drones offer a rapid, targeted collection of water samples from virtually anywhere on a lake with an active HAB without the need for a boat which can disturb the surrounding water. Drones are, however, limited in their ability to operate during inclement weather such as rain and heavy winds. Collectively, our results highlight numerous opportunities for drone-based water sampling technologies to track, predict, and respond to HABs in the future

Drone-based water sampling and characterization of three freshwater harmful algal blooms in the United States

Freshwater harmful algal blooms (HABs), caused mostly by toxic cyanobacteria, produce a range of cyanotoxins that threaten the health of humans and domestic animals. Climate conditions and anthropogenic influences such as agricultural run-off can alter the onset and intensity of HABs. Little is known about the distribution and spread of freshwater HABs. Current sampling protocols in some lakes involve teams of researchers that collect samples by hand from a boat and/or from the shoreline. Water samples can be collected from the surface, from discrete-depth collections, and/or from depth-integrated intervals. These collections are often restricted to certain months of the year, and generally are only performed at a limited number of collection sites. In lakes with active HABs, surface samples are generally sufficient for HAB water quality assessments. We used a unique DrOne Water Sampling SystEm (DOWSE) to collect water samples from the surface of three different HABs in Ohio (Grand Lake St Marys, GLSM and Lake Erie) and Virginia (Lake Anna), United States in 2019. The DOWSE consisted of a 3D-printed sampling device tethered to a drone (uncrewed aerial system, or UAS), and was used to collect surface water samples at different distances (10–100 m) from the shore or from an anchored boat. One hundred and eighty water samples (40 at GLSM, 20 at Lake Erie, and 120 at Lake Anna) were collected and analyzed from 18 drone flights. Our methods included testing for cyanotoxins, phycocyanin, and nutrients from surface water samples. Mean concentrations of microcystins (MCs) in drone water samples were 15.00, 1.92, and 0.02 ppb for GLSM, Lake Erie, and Lake Anna, respectively. Lake Anna had low levels of anatoxin in nearly all (111/120) of the drone water samples. Mean concentrations of phycocyanin in drone water samples were 687, 38, and 62 ppb for GLSM, Lake Erie, and Lake Anna, respectively. High levels of total phosphorus were observed in the drone water samples from GLSM (mean of 0.34 mg/L) and Lake Erie (mean of 0.12 mg/L). Lake Anna had the highest variability of total phosphorus with concentrations that ranged from 0.01 mg/L to 0.21 mg/L, with a mean of 0.06 mg/L. Nitrate levels varied greatly across sites, inverse with bloom biomass, ranging from below detection to 3.64 mg/L, with highest mean values in Lake Erie followed by GLSM and Lake Anna, respectively. Drones offer a rapid, targeted collection of water samples from virtually anywhere on a lake with an active HAB without the need for a boat which can disturb the surrounding water. Drones are, however, limited in their ability to operate during inclement weather such as rain and heavy winds. Collectively, our results highlight numerous opportunities for drone-based water sampling technologies to track, predict, and respond to HABs in the future

Activated Platelets in Carotid Artery Thrombosis in Mice Can Be Selectively Targeted with a Radiolabeled Single-Chain Antibody

BACKGROUND: Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis. METHODOLOGY/PRINCIPAL FINDINGS: LIBS as well as an unspecific control single-chain antibody were labeled with (111)Indium ((111)In) via bifunctional DTPA ( = (111)In-LIBS/(111)In-control). Autoradiography after incubation with (111)In-LIBS on activated platelets in vitro (mean 3866 ± 28 DLU/mm(2), 4010 ± 630 DLU/mm(2) and 4520 ± 293 DLU/mm(2)) produced a significantly higher ligand uptake compared to (111)In-control (2101 ± 76 DLU/mm(2), 1181 ± 96 DLU/mm(2) and 1866 ± 246 DLU/mm(2)) indicating a specific binding to activated platelets; P<0.05. Applying these findings to an ex vivo mouse model of carotid artery thrombosis revealed a significant increase in ligand uptake after injection of (111)In-LIBS in the presence of small thrombi compared to the non-injured side, as confirmed by histology (49630 ± 10650 DLU/mm(2) vs. 17390 ± 7470 DLU/mm(2); P<0.05). These findings could also be reproduced in vivo. SPECT-CT analysis of the injured carotid artery with (111)In-LIBS resulted in a significant increase of the target-to-background ratio compared to (111)In-control (1.99 ± 0.36 vs. 1.1 ± 0.24; P < 0.01). CONCLUSIONS/SIGNIFICANCE: Nuclear imaging with (111)In-LIBS allows the detection of platelet activation in vitro and ex vivo with high sensitivity. Using SPECT-CT, wall-adherent activated platelets in carotid arteries could be depicted in vivo. These results encourage further studies elucidating the role of activated platelets in plaque pathology and atherosclerosis and might be of interest for further developments towards clinical application

The Economic, Social and Administrative Pharmacy (ESAP) Discipline in US Schools and Colleges of Pharmacy

Schools and colleges of pharmacy in the United States have struggled over the past several decades with identifying a consistent title for the broad body of knowledge related to the social, economic, behavioral, and administrative aspects of pharmacy. This paper examines the educational background and professional experience of those teaching Economic, Social, Administrative Pharmacy (ESAP) content in the United States, to identify the type, frequency, and extent to which ESAP courses are taught in United State’s schools and colleges of pharmacy curricula, and to investigate the perceived importance of ESAP content among ESAP faculty. The data was collected by using a 23-item questionnaire was sent via email to 225 ESAP pharmacy faculties in the United States. 96 were returned completed for a 48 percent response rate. Most respondents are not housed in a standalone ESAP department, but would like to be. Both the educational backgrounds of ESAP faculty and the content taught within the discipline vary greatly. This may be because the content within the ESAP discipline is extremely broad. In addition, the diversification in content may be a result of both the diversity of instructor’s educational backgrounds and the lack of a generally approved definition for the ESAP field

Chemiluminescence in activated human neutrophils

Human neutrophils (PMNs) suspended in Hanks' balanced salt solution (HBSS), which are stimulated either by polycation-opsonized streptococci or by phorbol myristate acetate (PMA), generate nonamplified (CL), luminol-dependent (LDCL), and lucigenin-dependent chemiluminescence (LUCDCL). Treatment of activated PMNs with azide yielded a very intense CL response, but only a small LDCL or LUCDCL responses, when horse radish peroxidase (HRP) was added. Both CL and LDCL depend on the generation of Superoxide and on myeloperoxidase (MPO). Treatment of PMNs with azide followed either by dimethylthiourea (DMTU), deferoxamine, EDTA, or detapac generated very little CL upon addition of HRP, suggesting that CL is the: result of the interaction among H 2 O 2 , a peroxidase, and trace metals. In a cell-free system practically no CL was generated when H 2 O 2 was mixed with HRP in distilled water (DW). On the other hand significant CL was generated when either HBSS or RPMI media was employed. In both cases CL was markedly depressed either by deferoxamine or by EDTA, suggesting that these media might be contaminated by trace metals, which catalyzed a Fenton-driven reaction. Both HEPES and Tris buffers, when added to DW, failed to support significant HRP-induced CL. Nitrilotriacetate (NTA) chelates of Mn 2+ , Fe 2+ , Cu 2+ , and Co 2+ very markedly enhanced CL induced by mixtures of H 2 O 2 and HRP when distilled water was the supporting medium. Both HEPES and Tris buffer when added to DW strongly quenced NTA-metal-catalyzed CL. None of the NTA-metal chelates could boost CL generation by activated PMNs, because the salts in HBSS and RPMI interfered with the activity of the added metals. CL and LDCL of activated PMNs was enhanced by aminotriazole, but strongly inhibited by diphenylene iodonium (an inhibitor of NADPH oxidase) by azide, sodium cyanide (CN), cimetidine, histidine, benzoate, DMTU and moderately by Superoxide dismutase (SOD) and by deferoxamine. LUCDCL was markedly inhibited only by SOD but was boosted by CN. Taken together, it is suggested that CL generated by stimulated PMNs might be the result of the interactions among, NADPH oxidase, (inhibitable by diphenylene iodonium), MPO (inhibitable by sodium azide), H 2 O 2 probably of intracellular origin (inhibitable by DMTU but not by catalase), and trace metals that contaminate salt solutions. The nature of the salt solutions employed to measure CL in activated PMNs is critical.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44510/1/10753_2004_Article_BF00918987.pd