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Resolution of inflammation is an important physiological process following infection or injury. When inflammation fails to resolve, it can cause chronic inflammation, which exacerbates a myriad of diseases. Current anti-inflammatory treatment options are often inadequate to resolve inflammation, and as such, a key goal for drug discovery is to find natural products and novel compounds that can target immune resolution processes. In order to efficiently discovery new therapies, immune cell lines are often used, in conjunction with flow cytometry, to quickly and inexpensively screen potential drugs for immunomodulatory effects. However, seemingly minor or trivial differences in methodology can lead to inconsistent results across experiments and across laboratories. It was the goal of this project to examine the effects of those differences on the RAW 264.7 macrophage cell line, particularly as it relates to macrophage polarization experimentation. We found that the type of detachment method when preparing cells for flow cytometry can alter several key macrophage parameters, including markers for macrophage polarization, depending on the gating strategy used in identifying sub-populations of cells for analysis. Investigators need to incorporate best-practices in gating strategy in order to target viable cells that are not in aggregate to ensure consistent and reliable results for immunomodulatory drug discovery.
Jennifer R. McCall; Kathryn T. Sausman. Systematic approach in macrophage polarization experiments: Maintaining integrity and reproducibility using flow cytometry and sample preparation. Journal of Immunological Methods 2021, 492, 112969 .
AMA StyleJennifer R. McCall, Kathryn T. Sausman. Systematic approach in macrophage polarization experiments: Maintaining integrity and reproducibility using flow cytometry and sample preparation. Journal of Immunological Methods. 2021; 492 ():112969.
Chicago/Turabian StyleJennifer R. McCall; Kathryn T. Sausman. 2021. "Systematic approach in macrophage polarization experiments: Maintaining integrity and reproducibility using flow cytometry and sample preparation." Journal of Immunological Methods 492, no. : 112969.
A new monogalactosyldiacylglycerol (MGDG), a known monogalactosylmonoacylglycerol (MGMG) and a known polyunsaturated fatty acid methyl ester (PUFAME) were isolated from the marine dinoflagellate Karenia mikimotoi. The planar structure of the glycolipids was elucidated using mass spectroscopy (MS) and nuclear magnetic resonance (NMR) analyses and comparisons to the known glycolipid to confirm its structure. The MGDG was characterized as 3-O-β-D-galactopyranosyl-1-O-3,6,9,12,15-octadecapentaenoyl-2-O-tetradecanoylglycerol 1. The MGMG and PUFAME were characterized as (2S)-3-O-β-D-galactopyranosyl-1-O-3,6,9,12,15-octadecapentaenoylglycerol 2 and Methyl (3Z,6Z,9Z,12Z,15Z)-octadeca-3,6,9,12,15-pentaenoate 3, respectively. The isolation of the PUFAME strongly supports the polyunsaturated fatty acid (PUFA) fragment of these glycolipids. The relative configuration of the sugar was deduced by comparisons of 3JHH values and proton chemical shifts with those of known glycolipids. All isolated compounds MGDG, MGMG and PUFAME 1-3 were evaluated for their antimicrobial and anti-inflammatory activity. All compounds modulated macrophage responses, with compound 3 exhibiting the greatest anti-inflammatory activity.
Alain S. Leutou; Jennifer R. McCall; Robert York; Rajeshwar R. Govindapur; Andrea J. Bourdelais. Anti-Inflammatory Activity of Glycolipids and a Polyunsaturated Fatty Acid Methyl Ester Isolated from the Marine Dinoflagellate Karenia mikimotoi. Marine Drugs 2020, 18, 138 .
AMA StyleAlain S. Leutou, Jennifer R. McCall, Robert York, Rajeshwar R. Govindapur, Andrea J. Bourdelais. Anti-Inflammatory Activity of Glycolipids and a Polyunsaturated Fatty Acid Methyl Ester Isolated from the Marine Dinoflagellate Karenia mikimotoi. Marine Drugs. 2020; 18 (3):138.
Chicago/Turabian StyleAlain S. Leutou; Jennifer R. McCall; Robert York; Rajeshwar R. Govindapur; Andrea J. Bourdelais. 2020. "Anti-Inflammatory Activity of Glycolipids and a Polyunsaturated Fatty Acid Methyl Ester Isolated from the Marine Dinoflagellate Karenia mikimotoi." Marine Drugs 18, no. 3: 138.
Paralytic shellfish poisoning (PSP) is precipitated by a family of toxins produced by harmful algae, which are consumed by filter-feeding and commercially popular shellfish. The toxins, including saxitoxin, neosaxitoxin, and gonyautoxins, accumulate in shellfish and cause intoxication when consumed by humans and animals. Symptoms can range from minor neurological dysfunction to respiratory distress and death. There are over 40 different chemical congeners of saxitoxin and its analogs, many of which are toxic and many of which have low toxicity or are non-toxic. This makes accurate toxicity assessment difficult and complicates decisions regarding whether or not shellfish are safe to consume. In this study, we describe a new antibody-based bioassay that is able to detect toxic congeners (saxitoxin, neosaxitoxin, and gonyautoxins) with little cross-reactivity with the low or non-toxic congeners (decarbamoylated or di-sulfated forms). The anti-saxitoxin antibody used in this assay detects saxitoxin and neosaxitoxin, the two most toxic congers equally well, but not the relatively highly toxic gonyautoxins. By incorporating an incubation step with L-cysteine, it is possible to convert a majority of the gonyautoxins present to saxitoxin and neosaxitoxin, which are readily detected. The assay is, therefore, capable of detecting the most toxic PSP congeners found in commercially relevant shellfish. The assay was validated against samples whose toxicity was determined using standard HPLC methods and yielded a strong linear agreement between the methods, with R2 values of 0.94–0.96. As ELISAs are rapid, inexpensive, and easy-to-use, this new commercially available PSP ELISA represents an advance in technology allowing better safety management of the seafood supply and the ability to screen large numbers of samples that can occur when monitoring is increased substantially in response to toxic bloom events
Jennifer R. McCall; W. Christopher Holland; Devon M. Keeler; D. Ransom Hardison; R. Wayne Litaker. Improved Accuracy of Saxitoxin Measurement Using an Optimized Enzyme-Linked Immunosorbent Assay. Toxins 2019, 11, 632 .
AMA StyleJennifer R. McCall, W. Christopher Holland, Devon M. Keeler, D. Ransom Hardison, R. Wayne Litaker. Improved Accuracy of Saxitoxin Measurement Using an Optimized Enzyme-Linked Immunosorbent Assay. Toxins. 2019; 11 (11):632.
Chicago/Turabian StyleJennifer R. McCall; W. Christopher Holland; Devon M. Keeler; D. Ransom Hardison; R. Wayne Litaker. 2019. "Improved Accuracy of Saxitoxin Measurement Using an Optimized Enzyme-Linked Immunosorbent Assay." Toxins 11, no. 11: 632.
Chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD), cystic fibrosis, and asthma, are some of the leading causes of illness and fatalities worldwide. The search for novel treatments led to the exploration of marine natural products as drug candidates to combat the debilitating effects of mucus accumulation and chronic inflammation. Previous research showed that an alga-derived compound, brevenal, could attenuate the effects of inflammatory agents, but the mechanisms by which it exerted its effects remained unclear. We investigated the effects of brevenal on lipopolysaccharide (LPS) induced cytokine/chemokine production from murine macrophages and human lung epithelial cells. It was found that brevenal reduces proinflammatory mediator secretion while preserving anti-inflammatory secretion from these cells. Furthermore, we found that brevenal does not alter cell surface Toll-like receptor 4 (TLR4) expression, thereby maintaining the cells’ ability to respond to bacterial infection. However, brevenal does alter macrophage activation states, as demonstrated by reduced expression of both M1 and M2 phenotype markers, indicating this putative anti-inflammatory drug shifts innate immune cells to a less active state. Such a mechanism of action would be ideal for reducing inflammation in the lung, especially with patients suffering from chronic respiratory diseases, where inflammation can be lethal.
Devon M. Keeler; Meghan K. Grandal; Jennifer R. McCall. Brevenal, a Marine Natural Product, is Anti-Inflammatory and an Immunomodulator of Macrophage and Lung Epithelial Cells. Marine Drugs 2019, 17, 184 .
AMA StyleDevon M. Keeler, Meghan K. Grandal, Jennifer R. McCall. Brevenal, a Marine Natural Product, is Anti-Inflammatory and an Immunomodulator of Macrophage and Lung Epithelial Cells. Marine Drugs. 2019; 17 (3):184.
Chicago/Turabian StyleDevon M. Keeler; Meghan K. Grandal; Jennifer R. McCall. 2019. "Brevenal, a Marine Natural Product, is Anti-Inflammatory and an Immunomodulator of Macrophage and Lung Epithelial Cells." Marine Drugs 17, no. 3: 184.
Brevetoxins are a family of ladder-framed polyether toxins produced during blooms of the marine dinoflagellate, Karenia brevis. Consumption of shellfish or finfish exposed to brevetoxins can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are believed to be due to the activation of voltage-sensitive sodium channels in cell membranes. The traditional cytotoxicity assay for detection of brevetoxins uses the Neuro-2A cell line, which must first be treated with the neurotoxins, ouabain and veratridine, in order to become sensitive to brevetoxins. In this study, we demonstrate several drawbacks of the Neuro-2A assay, which include variability for the EC50 values for brevetoxin and non-linear triphasic dose response curves. Ouabain/veratridine-treated Neuro-2A cells do not show a typical sigmoidal dose response curve in response to brevetoxin, but rather, have a polynomial shaped curve, which makes calculating EC50 values highly variable. We describe a new fluorescence live cell imaging model, which allows for accurate calculation of cytotoxicity via nuclear staining and additional measurement of other viability parameters depending on which aspect of the cell is stained. In addition, the SJCRH30 cell line shows promise as an alternative to Neuro-2A cells for testing brevetoxins without the need for ouabain and veratridine.
Jennifer R. McCall; Elizabeth A. Elliott; Andrea J. Bourdelais. A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy. Marine Drugs 2014, 12, 4868 -4882.
AMA StyleJennifer R. McCall, Elizabeth A. Elliott, Andrea J. Bourdelais. A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy. Marine Drugs. 2014; 12 (9):4868-4882.
Chicago/Turabian StyleJennifer R. McCall; Elizabeth A. Elliott; Andrea J. Bourdelais. 2014. "A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy." Marine Drugs 12, no. 9: 4868-4882.
The marine dinoflagellate Karenia brevis produces a family of neurotoxins known as brevetoxins. Brevetoxins elicit their effects by binding to and activating voltage-sensitive sodium channels (VSSCs) in cell membranes. K. brevis also produces brevenal, a brevetoxin antagonist, which is able to inhibit and/or negate many of the detrimental effects of brevetoxins. Brevenal binding to VSSCs has yet to be fully characterized, in part due to the difficulty and expense of current techniques. In this study, we have developed a novel fluorescence binding assay for the brevenal binding site. Several fluorescent compounds were conjugated to brevenal to assess their effects on brevenal binding. The assay was validated against the radioligand assay for the brevenal binding site and yielded comparable equilibrium inhibition constants. The fluorescence-based assay was shown to be quicker and far less expensive and did not generate radioactive waste or need facilities for handling radioactive materials. In-depth studies using the brevenal conjugates showed that, while brevenal conjugates do bind to a binding site in the VSSC protein complex, they are not displaced by known VSSC site specific ligands. As such, brevenal elicits its action through a novel mechanism and/or currently unknown receptor site on VSSCs.
Jennifer R. McCall; Allan J. Goodman; Henry M. Jacocks; Alysha M. Thompson; Daniel G. Baden; Andrea J. Bourdelais. Development of a Fluorescence Assay for the Characterization of Brevenal Binding to Rat Brain Synaptosomes. Journal of Natural Products 2014, 77, 2014 -2020.
AMA StyleJennifer R. McCall, Allan J. Goodman, Henry M. Jacocks, Alysha M. Thompson, Daniel G. Baden, Andrea J. Bourdelais. Development of a Fluorescence Assay for the Characterization of Brevenal Binding to Rat Brain Synaptosomes. Journal of Natural Products. 2014; 77 (9):2014-2020.
Chicago/Turabian StyleJennifer R. McCall; Allan J. Goodman; Henry M. Jacocks; Alysha M. Thompson; Daniel G. Baden; Andrea J. Bourdelais. 2014. "Development of a Fluorescence Assay for the Characterization of Brevenal Binding to Rat Brain Synaptosomes." Journal of Natural Products 77, no. 9: 2014-2020.
Brevenal is a ladder frame polyether produced by the dinoflagellate Karenia brevis. This organism is also responsible for the production of the neurotoxic compounds known as brevetoxins. Ingestion or inhalation of the brevetoxins leads to adverse effects such as gastrointestinal maladies and bronchoconstriction. Brevenal shows antagonistic behavior to the brevetoxins and shows beneficial attributes when administered alone. For example, in an asthmatic sheep model, brevenal has been shown to increase tracheal mucosal velocity, an attribute which has led to its development as a potential treatment for Cystic Fibrosis. The mechanism of action of brevenal is poorly understood and the exact binding site has not been elucidated. In an attempt to further understand the mechanism of action of brevenal and potentially develop a second generation drug candidate, a series of brevenal derivatives were prepared through modification of the aldehyde moiety. These derivatives include aliphatic, aromatic and heteroaromatic hydrazide derivatives. The brevenal derivatives were tested using in vitro synaptosome binding assays to determine the ability of the compounds to displace brevetoxin and brevenal from their native receptors. A sheep inhalation model was used to determine if instillation of the brevenal derivatives resulted in bronchoconstriction. Only small modifications were tolerated, with larger moieties leading to loss of affinity for the brevenal receptor and bronchoconstriction in the sheep model.
Allan Goodman; Jennifer R. McCall; Henry M. Jacocks; Alysha Thompson; Daniel Baden; William M. Abraham; Andrea Bourdelais. Structure Activity Relationship of Brevenal Hydrazide Derivatives. Marine Drugs 2014, 12, 1839 -1858.
AMA StyleAllan Goodman, Jennifer R. McCall, Henry M. Jacocks, Alysha Thompson, Daniel Baden, William M. Abraham, Andrea Bourdelais. Structure Activity Relationship of Brevenal Hydrazide Derivatives. Marine Drugs. 2014; 12 (4):1839-1858.
Chicago/Turabian StyleAllan Goodman; Jennifer R. McCall; Henry M. Jacocks; Alysha Thompson; Daniel Baden; William M. Abraham; Andrea Bourdelais. 2014. "Structure Activity Relationship of Brevenal Hydrazide Derivatives." Marine Drugs 12, no. 4: 1839-1858.
Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in beach goers. Consumption of either shellfish or finfish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of brevetoxin analogs and competitors to site 5 on these channels has historically been measured using a radioligand competition assay that is fraught with difficulty, including slow analysis time, production of radioactive waste, and cumbersome and expensive methods associated with the generation of radioactive labeled ligands. In this study, we describe the development of a novel fluorescent synaptosome binding assay for the brevetoxin receptor. BODIPY®-conjugated to PbTx-2 was used as the labeled ligand. The BODIPY®–PbTx-2 conjugate was found to displace [3H]–PbTx-3 from its binding site on VSSCs on rat brain synaptosomes with an equilibrium inhibition constant of 0.11 nM. We have shown that brevetoxin A and B analogs are all able to compete for binding with the fluorescent ligand. Most importantly, this assay was validated against the current site 5 receptor binding assay standard, the radioligand receptor assay for the brevetoxin receptor using [3H]–PbTx-3 as the labeled ligand. The fluorescence based assay yielded equilibrium inhibition constants comparable to the radioligand assay for all brevetoxin analogs. The fluorescence based assay was quicker, far less expensive, and did not generate radioactive waste or need radioactive facilities. As such, this fluorescence-based assay can be used to replace the current radioligand assay for site 5 on voltage-sensitive sodium channels and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.
Jennifer R. McCall; Henry M. Jacocks; Daniel G. Baden; Andrea J. Bourdelais. Development of a competitive fluorescence-based synaptosome binding assay for brevetoxins. Harmful Algae 2012, 19, 85 -91.
AMA StyleJennifer R. McCall, Henry M. Jacocks, Daniel G. Baden, Andrea J. Bourdelais. Development of a competitive fluorescence-based synaptosome binding assay for brevetoxins. Harmful Algae. 2012; 19 ():85-91.
Chicago/Turabian StyleJennifer R. McCall; Henry M. Jacocks; Daniel G. Baden; Andrea J. Bourdelais. 2012. "Development of a competitive fluorescence-based synaptosome binding assay for brevetoxins." Harmful Algae 19, no. : 85-91.