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1.
Med Vet Entomol ; 37(1): 37-46, 2023 03.
Article in English | MEDLINE | ID: mdl-36054704

ABSTRACT

The beef industry endures major economic losses from a complex of flies that feed on bovine blood and mucus. For cattle on pasture, the most important of these pests are horn flies (Haematobia irritans [L.] [Diptera: Muscidae]) and face flies (Musca autumnalis [Diptera: Muscidae] De Geer). Pasture dragging to spread manure pats has been promoted as a management tactic for these species because their larvae inhabit bovine manure pats, but the efficacy of this practice has not been empirically validated. Spreading pats might promote fly mortality through desiccation or overheating, but these processes are weather-dependent and warrant testing in disparate climates. We evaluated pasture dragging effects while monitoring for weather interactions throughout nine experiment rounds in summers of 2018 and 2020 in Pennsylvania, USA. The manure spreading treatments increased pat surface area up to 300% but failed to significantly reduce emergence of horn flies and face flies as compared to controls. In contrast, precipitation and temperature were significant predictors in fly emergence models. Surprisingly, face fly emergence was significantly elevated in dragged pats twice in 2020. These data call for a reevaluation of pasture dragging as a management technique for horn flies and face flies across a range of climates.


Subject(s)
Muscidae , Animals , Cattle , Manure , Seasons , Feces , North America
2.
Acta Biomater ; 149: 198-212, 2022 09 01.
Article in English | MEDLINE | ID: mdl-35809788

ABSTRACT

Therapeutic benefits of curcumin for inflammatory diseases have been demonstrated. However, curcumin's potential as a clinical therapeutic has been hindered due to its low solubility and stability in vivo. We hypothesized that a hybrid curcumin carrier that incorporates albumin-binding and extracellular vesicle (EV) encapsulation could effectively address the current challenges of curcumin delivery. We further postulated that using dissolvable microneedle arrays (dMNAs) for local delivery of curcumin-albumin-EVs (CA-EVs) could effectively control skin inflammation in vivo. Mild sonication was used to encapsulate curcumin and albumin into EVs, and the resulting CA-EVs were integrated into tip-loaded dMNAs. In vitro and in vivo studies were performed to assess the stability, cellular uptake, and anti-inflammatory bioactivity of dMNA-delivered CA-EVs. Curcumin in CA-EVs exhibited at least five-fold higher stability in vitro than naïve curcumin or curcumin-EVs without albumin. Incorporating CA-EVs into dMNAs did not alter their cellular uptake or anti-inflammatory bioactivity. The dMNA embedded CA-EVs retained their bioactivity when stored at room temperature for at least 12 months. In rat and mice models, dMNA delivered CA-EVs suppressed and significantly reduced lipopolysaccharide and Imiquimod-triggered inflammation. We conclude that dMNA delivery of CA-EVs has the potential to become an effective local-delivery strategy for inflammatory skin diseases. STATEMENT OF SIGNIFICANCE: We introduce and evaluate a skin-targeted delivery system for curcumin that synergistically combines albumin association, extracellular-vesicle encapsulation, and dissolvable microneedle arrays (dMNAs) . In vitro, curcumin-albumin encapsulated extracellular vesicles (CA-EVs) inhibit and reverse the LPS-triggered expression of inflammatory transcription factor NF-κB. The integration of CA-EVs into dMNAs does not affect them physically or functionally. Importantly, dMNAs extend EV storage stability for at least 12 months at room temperature with minimal loss in their bioactivity. We demonstrate that dMNA delivered CA-EVs effectively block and reverse skin inflammation in vivo in mouse and rat models.


Subject(s)
Curcumin , Extracellular Vesicles , Albumins/pharmacology , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Curcumin/pharmacology , Inflammation/drug therapy , Mice , Rats
3.
Plant Cell Environ ; 44(4): 1142-1150, 2021 04.
Article in English | MEDLINE | ID: mdl-33277710

ABSTRACT

Sunlight filtered by green plant tissue becomes diminished in its ratio of red to far-red wavelengths (R:FR). Some parasitic plants exploit this change by growing towards regions of low R:FR to locate host plants. In principle, variation in R:FR can also convey ecologically relevant information about host proximity or architecture. Here, we demonstrate that the parasitic vine Cuscuta epilinum Weihe (Convolvulaceae) can distinguish fine-scale differences in R:FR associated with differences in the proximity and shape of potential host plants. We conducted dual-choice experiments by placing parasite seedlings between targets, including low R:FR fields manipulated via LED lighting and pairs of model plants exhibiting realistic R and FR reflectance but differing in proximity or shape. Seedlings consistently distinguished between low-R:FR fields of differing intensity. Furthermore, they exhibited preferences for nearer plant models versus identical models placed 4 cm further away and between same-sized models exhibiting shape differences. Our results indicate that parasites can discriminate minute differences in R:FR signatures corresponding to host factors (proximity and shape) that impact seedling survival. This keen sensory ability underpins the parasite's sophisticated foraging behaviour and highlights the broader importance of light cues in plant ecology.


Subject(s)
Cuscuta/physiology , Cuscuta/growth & development , Cuscuta/radiation effects , Flax/anatomy & histology , Flax/parasitology , Light , Solanum lycopersicum/anatomy & histology , Solanum lycopersicum/parasitology , Plants/anatomy & histology , Plants/parasitology
4.
Wound Repair Regen ; 27(2): 139-149, 2019 03.
Article in English | MEDLINE | ID: mdl-30576033

ABSTRACT

Cutaneous wounds caused by an exposure to high doses of ionizing radiation remain a therapeutic challenge. While new experimental strategies for treatment are being developed, there are currently no off-the-shelf therapies for the treatment of cutaneous radiation injury that have been proven to promote repair of the damaged tissues. Plasma-based biomaterials are biologically active biomaterials made from platelet enriched plasma, which can be made into both solid and semi-solid forms, are inexpensive, and are available as off-the-shelf, nonrefrigerated products. In this study, the use of plasma-based biomaterials for the mitigation of acute and late toxicity for cutaneous radiation injury was investigated using a mouse model. A 2-cm diameter circle of the dorsal skin was irradiated with a single dose of 35 Gy followed by topical treatment with plasma-based biomaterial or vehicle once daily for 5 weeks postirradiation. Weekly imaging demonstrated more complete wound resolution in the plasma-based biomaterial vs. vehicle group which became statistically significant (p < 0.05) at weeks 12, 13, and 14 postmaximum wound area. Despite more complete wound healing, at 9 and 17 weeks postirradiation, there was no statistically significant difference in collagen deposition or skin thickness between the plasma-based biomaterial and vehicle groups based on Masson trichrome staining nor was there a statistically significant difference in inflammatory or fibrosis-related gene expression between the groups. Although significant improvement was not observed for late toxicity, plasma-based biomaterials were effective at promoting wound closure, thus helping to mitigate acute toxicity.


Subject(s)
Biocompatible Materials/therapeutic use , Platelet-Rich Plasma , Radiation Injuries/pathology , Radiation Injuries/therapy , Skin/pathology , Animals , Biocompatible Materials/pharmacology , Cost-Benefit Analysis , Disease Models, Animal , Male , Mice , Wound Healing
5.
Int J Surg Case Rep ; 41: 269-271, 2017.
Article in English | MEDLINE | ID: mdl-29121583

ABSTRACT

INTRODUCTION: Lymphocytic phlebitis is a benign condition characterised by inflammation of the veins and rarely affects the gastrointestinal tract. Reported cases present as acute abdomen and involve the colon or small intestine. We report the fourth case of gastric lymphocytic phlebitis in the literature. PRESENTATION OF CASE: A 74-year-old female presented with eight weeks of abdominal pain. Findings at endoscopy were suggestive of a malignant ulcer on the greater curvature of antrum, while biopsies showed chronic gastritis without malignancy. Appearance at diagnostic laparoscopy was consistent with a malignant gastric ulcer with serosal changes. Due to persistent pain and the macroscopic appearance, she proceeded to have an open subtotal gastrectomy and D2 lymph node clearance. Despite macroscopic appearance, the microscopic examination demonstrated no malignancy, and was consistent with lymphocytic phlebitis with overlying ulceration. DISCUSSION: This case was a mimic for gastric malignancy, with the benign diagnosis only being made after surgical resection. Gastric lymphocytic phlebitis is a rare differential diagnosis for gastric ulcers when biopsies are negative, although preoperative diagnosis is difficult given the lesions do not involve the mucosa. CONCLUSION: If clinical history and endoscopic findings are suspicious for malignancy, despite normal biopsies, an aggressive surgical resection remains reasonable given the rarity gastric lymphocytic phlebitis.

6.
Proc Biol Sci ; 284(1849)2017 02 22.
Article in English | MEDLINE | ID: mdl-28228510

ABSTRACT

Plant trichomes constitute a first line of defence against insect herbivores. The pre- and post-ingestive defensive functions of glandular trichomes are well documented and include direct toxicity, adhesion, antinutrition and defence gene induction. By contrast, the defensive functions of non-glandular trichomes are less well characterized, although these structures are thought to serve as physical barriers that impede herbivore feeding and movement. We experimentally varied the density of stellate non-glandular trichomes in several ways to explore their pre- and post-ingestive effects on herbivores. Larvae of Manduca sexta (Sphingidae) initiated feeding faster and gained more weight on Solanum carolinense (Solanaceae) leaves having lower trichome densities (or experimentally removed trichomes) than on leaves having higher trichome densities. Adding trichomes to artificial diet also deterred feeding and adversely affected caterpillar growth relative to controls. Scanning electron and light microscopy revealed that the ingestion of stellate trichomes by M. sexta caterpillars caused extensive damage to the peritrophic membrane, a gut lining that is essential to digestion and pathogen isolation. These findings suggest that, in addition to acting as a physical barrier to deter feeding, trichomes can inhibit caterpillar growth and development via post-ingestive effects.


Subject(s)
Herbivory , Manduca , Plant Leaves/anatomy & histology , Solanum , Trichomes/anatomy & histology , Animals , Digestive System/pathology
7.
Plant Physiol ; 172(1): 181-97, 2016 09.
Article in English | MEDLINE | ID: mdl-27482077

ABSTRACT

Parasitic plants acquire diverse secondary metabolites from their hosts, including defense compounds that target insect herbivores. However, the ecological implications of this phenomenon, including the potential enhancement of parasite defenses, remain largely unexplored. We studied the translocation of glucosinolates from the brassicaceous host plant Arabidopsis (Arabidopsis thaliana) into parasitic dodder vines (Convolvulaceae; Cuscuta gronovii) and its effects on the parasite itself and on dodder-aphid interactions. Aliphatic and indole glucosinolates reached concentrations in parasite tissues higher than those observed in corresponding host tissues. Dodder growth was enhanced on cyp79B2 cyp79B3 hosts (without indole glucosinolates) but inhibited on atr1D hosts (with elevated indole glucosinolates) relative to wild-type hosts, which responded to parasitism with localized elevation of indole and aliphatic glucosinolates. These findings implicate indole glucosinolates in defense against parasitic plants. Rates of settling and survival on dodder vines by pea aphids (Acyrthosiphon pisum) were reduced significantly when dodder parasitized glucosinolate-producing hosts (wild type and atr1D) compared with glucosinolate-free hosts (cyp79B2 cyp79B3 myb28 myb29). However, settling and survival of green peach aphids (Myzus persicae) were not affected. M. persicae population growth was actually reduced on dodder parasitizing glucosinolate-free hosts compared with wild-type or atr1D hosts, even though stems of the former contain less glucosinolates and more amino acids. Strikingly, this effect was reversed when the aphids fed directly upon Arabidopsis, which indicates an interactive effect of parasite and host genotype on M. persicae that stems from host effects on dodder. Thus, our findings indicate that glucosinolates may have both direct and indirect effects on dodder-feeding herbivores.


Subject(s)
Aphids/physiology , Arabidopsis/metabolism , Cuscuta/physiology , Glucosinolates/metabolism , Animals , Aphids/metabolism , Arabidopsis/genetics , Arabidopsis/parasitology , Chromatography, High Pressure Liquid , Cuscuta/metabolism , Cuscuta/parasitology , Feeding Behavior/physiology , Genotype , Host-Parasite Interactions , Indoles/metabolism , Mutation
8.
Innovations (Phila) ; 11(1): 59-63; discussion 63, 2016.
Article in English | MEDLINE | ID: mdl-26918312

ABSTRACT

OBJECTIVE: This study aimed to test the impact of a plasma-based, material (PBM) impregnated with amiodarone on atrial electrophysiology and atrial fibrillation susceptibility in a porcine post-cardiac surgery model. METHODS: Ten healthy pigs underwent implantation of transvenous pacing systems, after which sterile talc was infused into the pericardial sac via a pericardiotomy. In five animals, PBM was applied to the atrial epicardial surface just before talc infusion. Electrophysiologic evaluations were performed using the pacing system immediately after chest closure and 7 days later. Atrial histologic evaluations were performed. RESULTS: Immediately after chest closure, there were no significant differences in electrophysiologic parameters between talc-only and talc + PBM animals, and atrial fibrillation was largely noninducible. On postsurgical day 7, electrophysiologic evaluation revealed significantly shorter sinus cycle length and atrioventricular nodal refractoriness among talc-only animals relative to talc + PBM animals, possibly suggesting attenuated sympathetic nervous system activation in the latter. Atrial fibrillation inducibility and duration were significantly greater among talc-only animals. No significant differences in atrial refractoriness or conduction time between groups were apparent. Histologic evaluation revealed a relative reduction in epicardial inflammation and less myolysis among talc + PBM animals. CONCLUSIONS: Epicardial application of a plasma-based, amiodarone-impregnated material was associated with a significant reduction in atrial inflammation and susceptibility to fibrillation.


Subject(s)
Amiodarone/administration & dosage , Atrial Fibrillation/prevention & control , Cardiac Pacing, Artificial/methods , Cardiac Surgical Procedures/methods , Animals , Cardiac Surgical Procedures/instrumentation , Disease Models, Animal , Heart Atria/drug effects , Male , Models, Animal , Swine , Talc/administration & dosage
9.
Biomaterials ; 60: 1-8, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25965281

ABSTRACT

Bacterial infection of subcutaneous "pockets" housing cardiovascular implantable electronic devices is a significant clinical complication. In this study, pacemakers encapsulated in a blood plasma-based material (PBM) composited with antibiotics were investigated for use as prophylactics against such infections. PBMs, which are made from pooled allogeneic plasma and platelets, are off-the-shelf biomaterials that can be manufactured in the form of complex 3D shapes, extrudable putties, or injectable pastes. In vitro studies with PBM pastes formulated with rifampicin and minocycline demonstrated antibiotic release over 6 days, activity against Escherichia coli, and reduced cytotoxic effects of the antibiotics on fibroblasts. The materials were also evaluated in vivo in a rabbit model in which pacemaker pockets were inoculated with methicillin-resistant Staphylococcus aureus (S. aureus) strain and examined 1 week later. The pockets containing the pacemaker plus S. aureus were grossly purulent and culture positive, whereas pockets into which PBM with antibiotics were injected around the pacemaker were free of purulence and culture negative (p < 0.001). None of the pockets into which PBM without antibiotics were placed demonstrated purulence, but 60% were culture positive. These results demonstrate the potential of PBMs to deliver antibiotics to diminish the incidence of pocket infections for pacemakers and other implantable devices.


Subject(s)
Anti-Bacterial Agents/administration & dosage , Drug Carriers/chemistry , Minocycline/administration & dosage , Pacemaker, Artificial/adverse effects , Plasma/chemistry , Prosthesis-Related Infections/prevention & control , Rifampin/administration & dosage , Animals , Anti-Bacterial Agents/therapeutic use , Biocompatible Materials/chemistry , Drug Delivery Systems , Escherichia coli/drug effects , Escherichia coli Infections/etiology , Escherichia coli Infections/prevention & control , Methicillin-Resistant Staphylococcus aureus/drug effects , Minocycline/therapeutic use , Prostheses and Implants/adverse effects , Prosthesis-Related Infections/etiology , Rabbits , Rifampin/therapeutic use , Staphylococcal Infections/etiology , Staphylococcal Infections/prevention & control
11.
Curr Opin Plant Biol ; 16(4): 464-72, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23870786

ABSTRACT

Parasitic plants--which make their living by extracting nutrients and other resources from other plants--are important components of many natural ecosystems; and some parasitic species are also devastating agricultural pests. To date, most research on plant parasitism has focused on nutrient transfer from host to parasite and the impacts of parasites on host plants. Far less work has addressed potential effects of the translocation of bioactive non-nutrient solutes-such as phytohormones, secondary metabolites, RNAs, and proteins-on the development and physiology of parasitic plants and on their subsequent interactions with other organisms such as insect herbivores. A growing number of recent studies document the transfer of such molecules from hosts to parasites and suggest that they may have significant impacts on parasite physiology and ecology. We review this literature and discuss potential implications for management and priorities for future research.


Subject(s)
Embryophyta/metabolism , Plant Growth Regulators/metabolism , Plant Proteins/metabolism , RNA, Plant/metabolism , Symbiosis , Biological Transport
12.
Bioconjug Chem ; 18(3): 695-701, 2007.
Article in English | MEDLINE | ID: mdl-17432824

ABSTRACT

The goal of this work was to demonstrate that aprotinin conjugated to fibrinogen could (1) maintain its function and (2) control fibrin degradation. Using the chick chorioallantoic membrane (CAM) assay, we found that blood vessels did not directly invade fibrin constructs containing immobilized fibroblast growth factor-2. Because the fibrin quickly degraded within approximately 5 days, we hypothesized that controlling fibrinolysis may improve direct blood vessel invasion. Aprotinin, a protease inhibitor typically added to slow fibrinolysis, is a small protein and can diffuse out of the gel resulting in the loss of fibrinolysis protection. Therefore, using a novel synthesis strategy, aprotinin and a fluorescent reporter, Cy3, were chemically conjugated to fibrinogen. In vitro microplate absorbance assays showed that the conjugated aprotinin was able to inhibit plasmin-mediated fibrin degradation and that its activity was comparable to equimolar levels of soluble, nonconjugated aprotinin. Additionally, we found that fibrinolysis rates could be tuned by varying the level of conjugated aprotinin within the gel. The conjugated aprotinin also demonstrated functionality in vivo. In the chick CAM assay, fibrin gels containing conjugated aprotinin were approximately 5 times larger than gels containing soluble aprotinin after 4 days. Also, in support of our hypothesis, we found that immobilized aprotinin within fibrin gels demonstrated substantial blood vessel invasion.


Subject(s)
Aprotinin/chemistry , Carbocyanines/chemistry , Fibrin/chemistry , Fibrinogen/analogs & derivatives , Serine Proteinase Inhibitors/chemistry , Tissue Engineering/methods , Animals , Aprotinin/chemical synthesis , Blood Vessels/drug effects , Carbocyanines/chemical synthesis , Chick Embryo , Chorioallantoic Membrane/blood supply , Chorioallantoic Membrane/chemistry , Fibrinogen/chemical synthesis , Fibrinogen/chemistry , Fibrinolysin/antagonists & inhibitors , Fibrinolysis , Gels/chemistry , Gels/pharmacology
13.
Microvasc Res ; 73(2): 75-83, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17070560

ABSTRACT

Quantum dots (QDs) are fluorescent semiconductor nanocrystals that possess a number of superior fluorescent properties compared to more established organic dyes and fluorescent proteins. As a result, QDs are being studied for use in a wide range of biological applications. We have examined QDs for one such application, visualization of blood vessels of the chick chorioallantoic membrane (CAM), a popular model for studying various aspects of blood vessel development including angiogenesis. Intravitally injected QDs were found to be biocompatible and were kept in circulation over the course of 4 days without any observed deleterious effects. QD vascular residence time was tunable through QD surface chemistry modification. We also found that use of QDs with higher emission wavelengths (>655 nm) virtually eliminated all chick-derived autofluorescence and improved depth-of-field imaging. QDs were compared to FITC-dextrans, a fluorescent dye commonly used for imaging CAM vessels. QDs were found to image vessels as well as or better than FITC-dextrans at 2-3 orders of magnitude lower concentration. We also demonstrated that QDs are fixable with low fluorescence loss and thus can be used in conjunction with histological processing for further sample analysis.


Subject(s)
Allantois/blood supply , Chorion/blood supply , Quantum Dots , Animals , Chick Embryo , Dextrans , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescent Dyes , Microscopy, Electron , Microscopy, Fluorescence , Neovascularization, Physiologic , Time Factors
14.
Biochim Biophys Acta ; 1752(1): 18-25, 2005 Aug 31.
Article in English | MEDLINE | ID: mdl-16112628

ABSTRACT

Efficient protein folding and trafficking are essential for high-level production of secretory proteins. Slow folding or misfolding of proteins can lead to secretory bottlenecks that reduce productivity. We previously examined the expression of a hyperthermophilic tetramer Pyrococcus furiosus beta-glucosidase in the yeast Saccharomyces cerevisiae. A secretory bottleneck was found in the endoplasmic reticulum, presumably due to beta-glucosidase misfolding. By increasing expression temperature from 30 degrees C up to 40 degrees C, secretion yields increased by as much as 440% per cell to greater than 100 mg/L at 37 degrees C. We examined the effect of temperature on beta-glucosidase folding and secretion and determined that increased expression temperature decreased intracellularly retained, insoluble beta-glucosidase. Likewise, stress on the cell caused by beta-glucosidase expression was found to be greatly reduced at 37 degrees C compared to 30 degrees C. Levels of the abundant endoplasmic reticulum chaperone, BiP, were relatively unchanged at these temperatures during heterologous expression. Using cycloheximide to inhibit new protein synthesis, we determined that the increase in secretion is likely due to the effect of temperature on the beta-glucosidase itself rather than the cell's response to elevated temperatures. We believe that this is the first evidence of in vivo effects of temperature on the secretion of hyperthermophilic proteins.


Subject(s)
Enzymes/metabolism , Pyrococcus furiosus/enzymology , beta-Glucosidase/metabolism , Endoplasmic Reticulum/enzymology , Hot Temperature , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Thermodynamics , beta-Glucosidase/chemistry
15.
Biotechnol Bioeng ; 85(3): 340-50, 2004 Feb 05.
Article in English | MEDLINE | ID: mdl-14748090

ABSTRACT

In eukaryotes, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). Many heterologous proteins are retained in the ER due to suboptimal folding conditions. We previously reported that heterologous secretion of Pyrococcus furiosus beta-glucosidase in Saccharomyces cerevisiae resulted in the accumulation of a large fraction of inactive beta-glucosidase in the ER. In this work, we determine the effect of introducing additional genes of ER-resident yeast proteins, Kar2p (binding protein [BiP]) and protein disulfide isomerase (PDI), on relieving this bottleneck. Single-copy expression of BiP and PDI worked synergistically to improve secretion by reverse similar 60%. In an effort to optimize BiP and PDI interactions, we created a library of beta-glucosidase expression strains that incorporated four combinations of constitutively or inducibly-expressed BiP and PDI genes integrated to random gene copynumbers in the yeast chromosome. Approximately 15% of the transformants screened had secretion level improvements higher than that seen with single BiP/PDI gene overexpression, and the highest secreting strain had threefold higher beta-glucosidase levels than the control. Nineteen of the improved strains were re-examined for beta-glucosidase secretion as well as BiP and PDI levels. Within the improved transformants BiP and PDI levels ranged sevenfold and tenfold over the control, respectively. Interestingly, increasing BiP levels decreased beta-glucosidase secretion, whereas increasing PDI levels increased beta-glucosidase secretion. The action of PDI was unexpected because beta-glucosidase is not a disulfide-bonded protein. We suggest that PDI may be acting in a chaperone-like capacity or possibly creating mixed disulfides with the beta-glucosidase's lone cysteine residue during the folding and assembly process.


Subject(s)
Carrier Proteins/metabolism , Protein Disulfide-Isomerases/metabolism , Pyrococcus furiosus/metabolism , Saccharomyces cerevisiae/metabolism , beta-Glucosidase/biosynthesis , Carrier Proteins/genetics , Enzyme Activation , Gene Dosage , Genetic Enhancement/methods , Protein Disulfide-Isomerases/genetics , Pyrococcus furiosus/genetics , Recombinant Proteins/biosynthesis , Saccharomyces cerevisiae/genetics , Transfection/methods , beta-Glucosidase/genetics
16.
Vaccine ; 21(27-30): 4348-58, 2003 Oct 01.
Article in English | MEDLINE | ID: mdl-14505917

ABSTRACT

Immunization with native fusion (F) protein from respiratory syncytial virus (RSV) adsorbed to alum adjuvant generates greater than fourfold rises in serum neutralizing antibody titers in approximately 50% of seropositive humans. Using BALB/c mice we demonstrate herein that enhanced neutralization titers and accelerated clearance of virus from the lungs after challenge are possible if the attachment (G) glycoprotein is added to F protein-based vaccines. We further reveal for the first time that polarized type 2 T cell responses and immunopathology associated with G protein are inhibited by adjuvants recognized by toll-like receptors (TLR). Co-formulation with compounds that targeted TLR-2, TLR-3, TLR-4, or TLR-9 elicited significantly diminished type 2 T cell responses that caused granulocytic inflammation and eosinophilia in the airways after challenge. These results were not observed with recombinant IL-12 or QS-21. The data are important for improving combination vaccines for RSV.


Subject(s)
Adjuvants, Immunologic/pharmacology , Membrane Glycoproteins/drug effects , Receptors, Cell Surface/drug effects , Respiratory Syncytial Virus, Human/immunology , T-Lymphocytes/immunology , Viral Envelope Proteins/immunology , Animals , CpG Islands/genetics , Cytokines/biosynthesis , Eosinophils/pathology , Female , Flow Cytometry , Granulocytes/pathology , Interferon-gamma/physiology , Interleukin-12/physiology , Lung/pathology , Membrane Glycoproteins/immunology , Mice , Mice, Inbred BALB C , Phenotype , Receptors, Cell Surface/immunology , Thrombopoietin , Toll-Like Receptor 2 , Toll-Like Receptor 3 , Toll-Like Receptor 4 , Toll-Like Receptors , Viral Plaque Assay
17.
Biotechnol Bioeng ; 79(7): 713-23, 2002 Sep 30.
Article in English | MEDLINE | ID: mdl-12209794

ABSTRACT

Archaeal enzymes have great potential for industrial use; however, expressing them in their natural hosts has proven challenging. Growth conditions for many archaea are beyond typical fermentation capabilities, and to compound the problem, archaea generally achieve much lower biomass yields than Escherichia coli or Saccharomyces cerevisiae. To determine whether a eukaryotic host, S. cerevisiae, would be a suitable alternative for archaeal protein production, we examined the expression of the tetrameric beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. We engineered the beta-glucosidase to facilitate secretion into the culture medium and have demonstrated the beta-glucosidase's secretion and activity. We determined the dependence of beta-glucosidase secretion on gene copy number and obtained a transformant capable of secreting approximately 10 mg/L in batch culture. All transformants retained large intracellular fractions of beta-glucosidase, indicative of an intracellular bottleneck. Cell fractionation by sucrose density centrifugation and immunofluorescence identified the endoplasmic reticulum as the secretion bottleneck. Preliminary evidence indicates that the cause of this bottleneck is misfolding of the monomeric beta-glucosidase, rather than tetrameric association. Expression at moderately elevated temperatures (between 30 and 40 degrees C) improved beta-glucosidase yields, suggesting that higher temperature expression may improve folding and secretion yields.


Subject(s)
Endoplasmic Reticulum/metabolism , Gene Expression Regulation, Fungal , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , beta-Glucosidase/genetics , beta-Glucosidase/metabolism , Amino Acid Sequence , Cells, Cultured , Cloning, Molecular , Endoplasmic Reticulum/genetics , Gene Dosage , Gene Expression Regulation, Archaeal , Gene Expression Regulation, Enzymologic , Molecular Sequence Data , Protein Engineering/methods , Pyrococcus furiosus/enzymology , Pyrococcus furiosus/genetics , Quality Control , Reproducibility of Results , Saccharomyces cerevisiae/classification , Sensitivity and Specificity , Species Specificity , Temperature , Transformation, Genetic , Yeasts/genetics , Yeasts/metabolism
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