Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 11 de 11
Filter
Add more filters










Publication year range
1.
PLoS One ; 17(8): e0272163, 2022.
Article in English | MEDLINE | ID: mdl-36001626

ABSTRACT

A previous report demonstrated the strong association between the presence of antibodies binding to an epitope region from SARS-CoV-2 nucleocapsid, termed Ep9, and COVID-19 disease severity. Patients with anti-Ep9 antibodies (Abs) had hallmarks of antigenic interference (AIN), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a prior infection was hypothesized to drive formation of suboptimal anti-Ep9 Abs in severe COVID-19 infections. This study identifies a putative primary antigen capable of stimulating production of cross-reactive, anti-Ep9 Abs. Binding assays with patient blood samples directly show cross-reactivity between Abs binding to Ep9 and only one bioinformatics-derived, homologous putative antigen, a sequence derived from the neuraminidase protein of H3N2 influenza A virus. This cross-reactive binding is highly influenza strain specific and sensitive to even single amino acid changes in epitope sequence. The neuraminidase protein is not present in the influenza vaccine, and the anti-Ep9 Abs likely resulted from the widespread influenza infection in 2014. Therefore, AIN from a previous infection could underlie some cases of COVID-19 disease severity.


Subject(s)
COVID-19 , Influenza Vaccines , Influenza, Human , Antibodies, Viral , Epitopes , Humans , Influenza A Virus, H3N2 Subtype , Influenza, Human/prevention & control , Neuraminidase , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
2.
Angew Chem Int Ed Engl ; 61(23): e202202021, 2022 06 07.
Article in English | MEDLINE | ID: mdl-35333430

ABSTRACT

Unlocking the potential of personalized medicine in point-of-care settings requires a new generation of biomarker and proteomic assays. Ideally, assays could inexpensively perform hundreds of quantitative protein measurements in parallel at the bedsides of patients. This goal greatly exceeds current capabilities. Furthermore, biomarker assays are often challenging to translate from benchtop to clinic due to difficulties achieving and assessing the necessary selectivity, sensitivity, and reproducibility. To address these challenges, we developed an efficient (<5 min), robust (comparatively lower CVs), and inexpensive (decreasing reagent use and cost by >70 %) immunoassay method. Specifically, the immunoblot membrane is dotted with the sample and then developed in a vortex fluidic device (VFD) reactor. All assay steps-blocking, binding, and washing-leverage the unique thin-film microfluidics of the VFD. The approach can accelerate direct, indirect, and sandwich immunoblot assays. The applications demonstrated include assays relevant to both the laboratory and the clinic.


Subject(s)
Microfluidics , Proteomics , Acceleration , Humans , Immunoassay , Reproducibility of Results
3.
bioRxiv ; 2021 Jun 09.
Article in English | MEDLINE | ID: mdl-34127968

ABSTRACT

A previous report demonstrated the strong association between the presence of antibodies binding to an epitope region from SARS-CoV-2 nucleocapsid, termed Ep9, and COVID-19 disease severity. Patients with anti-Ep9 antibodies (Abs) had hallmarks of antigenic imprinting (AIM), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a previous infection was hypothesized to drive formation of suboptimal anti-Ep9 Abs in severe COVID-19 infections. This study identifies a putative primary antigen capable of stimulating production of cross-reactive, anti-Ep9 Abs. Binding assays with patient blood samples directly show cross-reactivity between Abs binding to Ep9 and only one bioinformatics-derived, homologous potential antigen, a sequence derived from the neuraminidase protein of H3N2 Influenza A virus. This cross-reactive binding is highly influenza strain specific and sensitive to even single amino acid changes in epitope sequence. The neuraminidase protein is not present in the influenza vaccine, and the anti-Ep9 Abs likely resulted from the widespread influenza infection in 2014. Therefore, AIM from a previous infection could underlie some cases of COVID-19 disease severity. IMPORTANCE: Infections with SARS-COV-2 result in diverse disease outcomes, ranging from asymptomatic to fatal. The mechanisms underlying different disease outcomes remain largely unexplained. Previously, our laboratory identified a strong association between the presence of an antibody and increased disease severity in a subset of COVID-19 patients. Here, we report that this severity-associated antibody cross-reacts with viral proteins from an influenza A viral strain from 2014. Therefore, we speculate that antibodies generated against previous infections, like the 2014 influenza A, play a significant role in directing some peoples’ immune responses against SARS-COV-2. Such understanding of the sources and drivers of COVID-19 disease severity can help early identification and pre-emptive treatment.

4.
mSphere ; 6(2)2021 04 28.
Article in English | MEDLINE | ID: mdl-33910993

ABSTRACT

Effective methods for predicting COVID-19 disease trajectories are urgently needed. Here, enzyme-linked immunosorbent assay (ELISA) and coronavirus antigen microarray (COVAM) analysis mapped antibody epitopes in the plasma of COVID-19 patients (n = 86) experiencing a wide range of disease states. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the intensive care unit (ICU), requirement for ventilators, or death. Importantly, anti-Ep9 antibodies can be detected within 6 days post-symptom onset and sometimes within 1 day. Furthermore, anti-Ep9 antibodies correlate with various comorbidities and hallmarks of immune hyperactivity. We introduce a simple-to-calculate, disease risk factor score to quantitate each patient's comorbidities and age. For patients with anti-Ep9 antibodies, scores above 3.0 predict more severe disease outcomes with a 13.42 likelihood ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic to allow early identification and triage of high-risk patients. Such information could guide more effective therapeutic intervention.IMPORTANCE The COVID-19 pandemic has resulted in over two million deaths worldwide. Despite efforts to fight the virus, the disease continues to overwhelm hospitals with severely ill patients. Diagnosis of COVID-19 is readily accomplished through a multitude of reliable testing platforms; however, prognostic prediction remains elusive. To this end, we identified a short epitope from the SARS-CoV-2 nucleocapsid protein and also a disease risk factor score based upon comorbidities and age. The presence of antibodies specifically binding to this epitope plus a score cutoff can predict severe COVID-19 outcomes with 96.7% specificity.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Severity of Illness Index , COVID-19/pathology , Cell Surface Display Techniques , Enzyme-Linked Immunosorbent Assay , Epitopes/blood , Epitopes/immunology , Humans , Nucleocapsid/immunology , Phosphoproteins/immunology , Prognosis , Risk Factors
5.
bioRxiv ; 2021 Mar 08.
Article in English | MEDLINE | ID: mdl-33083803

ABSTRACT

Effective methods for predicting COVID-19 disease trajectories are urgently needed. Here, ELISA and coronavirus antigen microarray (COVAM) analysis mapped antibody epitopes in the plasma of COVID-19 patients (n = 86) experiencing a wide-range of disease states. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the ICU, requirement for ventilators, or death. Importantly, anti-Ep9 antibodies can be detected within six days post-symptom onset and sometimes within one day. Furthermore, anti-Ep9 antibodies correlate with various comorbidities and hallmarks of immune hyperactivity. We introduce a simple-to-calculate, disease risk factor score to quantitate each patients comorbidities and age. For patients with anti-Ep9 antibodies, scores above 3.0 predict more severe disease outcomes with a 13.42 Likelihood Ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic to allow early identification and triage of high-risk patients. Such information could guide more effective therapeutic intervention.

6.
Anal Chem ; 92(11): 7683-7689, 2020 06 02.
Article in English | MEDLINE | ID: mdl-32352281

ABSTRACT

Molecular sensors from protein engineering offer new methods to sensitively bind to and detect target analytes for a wide range of applications. For example, these sensors can be integrated into probes for implantation, and then yield new and valuable physiological information. Here, a new Förster resonance energy transfer (FRET)-based sensor is integrated with an optical fiber to yield a device measuring free Ca2+. This membrane encapsulated optical fiber (MEOF) device is composed of a sensor matrix that fills poly(tetrafluoroethylene) (PTFE) with an engineered troponin C (TnC) protein fused to a pair of FRET fluorophores. The FRET efficiency is modulated upon Ca2+ ion binding. The probe further comprises a second, size-excluding filter membrane that is synthesized by filling the pores of a PTFE matrix with a poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogel; this design ensures protection from circulating proteases and the foreign body response. The two membranes are stacked and placed on a thin, silica optical fiber for optical excitation and detection. Results show the biosensor responds to changes in Ca2+ concentration within minutes with a sensitivity ranging from 0.01 to 10 mM Ca2+, allowing discrimination of hyper- and hypocalcemia. Furthermore, the system reversibly binds Ca2+ to allow continuous monitoring. This work paves the way for the use of engineered structure-switching proteins for continuous optical monitoring in a large number of applications.


Subject(s)
Calcium/analysis , Fluorescence Resonance Energy Transfer , Troponin C/metabolism , Animals , Batrachoidiformes/metabolism , Calcium/metabolism , Photochemical Processes , Proteolysis , Troponin C/chemistry
7.
Bioconjug Chem ; 31(5): 1449-1462, 2020 05 20.
Article in English | MEDLINE | ID: mdl-32302483

ABSTRACT

Advances in bioconjugation, the ability to link biomolecules to each other, small molecules, surfaces, and more, can spur the development of advanced materials and therapeutics. We have discovered that pyrocinchonimide, the dimethylated analogue of maleimide, undergoes a surprising transformation with biomolecules. The reaction targets amines and involves an imide transfer, which has not been previously reported for bioconjugation purposes. Despite their similarity to maleimides, pyrocinchonimides do not react with free thiols. Though both lysine residues and the N-termini of proteins can receive the transferred imide, the reaction also exhibits a marked preference for certain amines that cannot solely be ascribed to solvent accessibility. This property is peculiar among amine-targeting reactions and can reduce combinatorial diversity when many available reactive amines are available, such as in the formation of antibody-drug conjugates. Unlike amides, the modification undergoes very slow reversion under high pH conditions. The reaction offers a thermodynamically controlled route to single or multiple modifications of proteins for a wide range of applications.


Subject(s)
Amines/chemistry , Imides/chemistry , Proteins/chemistry , Hydrogen-Ion Concentration , Kinetics , Lysine/chemistry , Solvents/chemistry , Sulfhydryl Compounds/chemistry , Thermodynamics
8.
Biochem J ; 469(1): 71-82, 2015 Jul 01.
Article in English | MEDLINE | ID: mdl-25915851

ABSTRACT

Adiponectin mediates anti-diabetic effects via increasing hepatic insulin sensitivity and direct metabolic effects. In the present study, we conducted a comprehensive and unbiased metabolomic profiling of liver tissue from AdKO (adiponectin-knockout) mice, with and without adiponectin supplementation, fed on an HFD (high-fat diet) to derive insight into the mechanisms and consequences of insulin resistance. Hepatic lipid accumulation and insulin resistance induced by the HFD were reduced by adiponectin. The HFD significantly altered levels of 147 metabolites, and bioinformatic analysis indicated that one of the most striking changes was the profile of increased lysophospholipids. These changes were largely corrected by adiponectin, at least in part via direct regulation of PLA2 (phospholipase A2) as palmitate-induced PLA2 activation was attenuated by adiponectin in primary hepatocytes. Notable decreases in several glycerolipids after the HFD were reversed by adiponectin, which also corrected elevations in several diacyglycerol and ceramide species. Our data also indicate that stimulation of ω-oxidation of fatty acids by the HFD is enhanced by adiponectin. In conclusion, this metabolomic profiling approach in AdKO mice identified important targets of adiponectin action, including PLA2, to regulate lysophospholipid metabolism and ω-oxidation of fatty acids.


Subject(s)
Adiponectin/metabolism , Hepatocytes/metabolism , Lipid Metabolism/physiology , Liver/metabolism , Lysophospholipids/metabolism , Metabolome/physiology , Adiponectin/genetics , Animals , Hepatocytes/cytology , Liver/cytology , Lysophospholipids/genetics , Metabolomics , Mice , Mice, Knockout , Phospholipases A2/genetics , Phospholipases A2/metabolism
9.
Am J Physiol Endocrinol Metab ; 304(2): E168-75, 2013 Jan 15.
Article in English | MEDLINE | ID: mdl-23132298

ABSTRACT

Numerous studies have shown that adiponectin confers antidiabetic effects via both insulin-like and insulin-sensitizing actions. The majority of adiponectin in circulation is derived from adipocytes; however, other tissues such as skeletal muscle can produce adiponectin. This study was designed to investigate the functional significance of adiponectin produced by skeletal muscle. We encapsulated the adiponectin gene in lipid-coated microspheres filled with octafluoropropane gas that were injected into the systemic circulation and destroyed within the microvasculature of skeletal muscle using ultrasound. We first demonstrated safe and successful targeting of luciferase and green fluorescent protein reporter genes to skeletal muscle using this approach and then confirmed efficient overexpression of adiponectin mRNA and oligomeric protein forms. Glucose tolerance test indicated that overexpression of adiponectin in skeletal muscle was able to improve glucose intolerance induced by feeding mice a high-fat diet (HFD), and this correlated with improved skeletal muscle insulin signaling. We then performed hyperinsulinemic-euglycemic clamp studies and demonstrated that adiponectin overexpression attenuated the decreases in glucose infusion rate, glucose disposal, and increase in glucose appearance induced by HFD. Ultrasound-targeted microbubble destruction (UTMD) delivery of adiponectin to skeletal muscle also enhanced serum adiponectin levels and improved hepatic insulin sensitivity. In conclusion, our data show that UTMD efficiently delivers adiponectin to skeletal muscle and that this improves insulin sensitivity and glucose homeostasis.


Subject(s)
Adiponectin/genetics , Gene Transfer Techniques , Glucose/metabolism , Insulin Resistance/genetics , Microbubbles , Muscle, Skeletal/metabolism , Adiponectin/metabolism , Animals , Genetic Therapy/methods , Glucose Clamp Technique , Glucose Intolerance/genetics , Glucose Intolerance/metabolism , Glucose Intolerance/therapy , Homeostasis/genetics , Homeostasis/physiology , Male , Mice , Mice, Inbred C57BL , Microbubbles/therapeutic use , Muscle, Skeletal/diagnostic imaging , Organ Specificity/genetics , Ultrasonics/methods , Ultrasonography
10.
Am J Pathol ; 180(2): 715-26, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22236491

ABSTRACT

Diabetes mellitus accelerates atherosclerotic progression, peripheral angiopathy development, and arterial hypertension, all of which are associated with elastic fiber disease. However, the potential mechanistic links between insulin deficiency and impaired elastogenesis in diabetes have not been explored. Results of the present study reveal that insulin administered in therapeutically relevant concentrations (0.5 to 10 nmol/L) selectively stimulates formation of new elastic fibers in cultures of human aortic smooth muscle cells. These concentrations of insulin neither up-regulate collagen type I and fibronectin deposition nor stimulate cellular proliferation. Further, the elastogenic effect of insulin occurs after insulin receptor activation, which triggers the PI3K downstream signaling pathway and activates elastin gene transcription. In addition, the promoter region of the human elastin gene contains the CAAATAA sequence, consistent with the FoxO-recognized element, and the genomic effects of insulin occur after removal of the FoxO1 transcriptional inhibitor from the FoxO-recognized element in the elastin gene promoter. In addition, insulin signaling facilitates the association of tropoelastin with its specific 67-kDa elastin-binding protein/spliced form of ß-galactosidase chaperone, enhancing secretion. These results are crucial to understanding of the molecular and cellular mechanisms of diabetes-associated vascular disease, and, in particular, endorse use of insulin therapy for treatment of atherosclerotic lesions in patients with type 1 diabetes, in which induction of new elastic fibers would mechanically stabilize the developing plaques and prevent arterial occlusions.


Subject(s)
Aorta/metabolism , Elastin/biosynthesis , Insulin/pharmacology , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Aorta/cytology , Cell Proliferation , Cells, Cultured , Child , Child, Preschool , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Galactosidases/metabolism , Humans , Male , Morpholines/pharmacology , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Phosphatidylinositol 3-Kinases/metabolism , RNA Splicing Factors , RNA-Binding Proteins/drug effects , Tropoelastin/metabolism , Up-Regulation
11.
J Biol Chem ; 286(42): 36580-91, 2011 Oct 21.
Article in English | MEDLINE | ID: mdl-21880723

ABSTRACT

The mechanism that leads to the inverse relationship between heightened cellular proliferation and the cessation of elastic fibers production, observed during formation of the arterial occlusions and dermal scars, is not fully understood. Because the retinoblastoma protein (Rb), responsible for cell cycle initiation, has also been implicated in insulin-like growth factor-I-mediated signaling stimulating elastin gene activation, we explored whether differential phosphorylation of Rb by various cyclin·cyclin-dependent kinase complexes would be responsible for promoting either elastogenic or pro-proliferative signals. We first tested cultures of dermal fibroblasts derived from Costello syndrome patients, in which heightened proliferation driven by mutated oncogenic H-Ras coincides with inhibition of elastogenesis. We found that Costello syndrome fibroblasts display elevated level of Rb phosphorylation on serine 780 (Ser(P)-780-Rb) and that pharmacological inhibition of Ras with radicicol, Mek/Erk with PD98059, or cyclin-dependent kinase 4 with PD0332991 not only leads to down-regulation of Ser(P)-780-Rb levels but also enhances Rb phosphorylation on threonine-821 (Thr(P)-821-Rb), which coincides with the recovery of elastin production. Then we demonstrated that treatment of normal skin fibroblasts with the pro-proliferative PDGF BB also up-regulates Ser(P)-780-Rb levels, but treatment with the pro-elastogenic insulin-like growth factor-I activates cyclinE-cdk2 complex to phosphorylate Rb on Thr-821. Importantly, we have established that elevation of Thr(P)-821-Rb promotes Rb binding to the Sp1 transcription factor and that successive binding of the Rb-Sp1 complex to the retinoblastoma control element within the elastin gene promoter stimulates tropoelastin transcription. In summary, we provide novel insight into the role of Rb in mediating the inverse relationship between elastogenesis and cellular proliferation.


Subject(s)
Cell Proliferation , Dermis/metabolism , Elastin/biosynthesis , Fibroblasts/metabolism , Retinoblastoma Protein/metabolism , Adolescent , Adult , Becaplermin , Cells, Cultured , Costello Syndrome , Dermis/pathology , Elastin/genetics , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Fibroblasts/pathology , Flavonoids/pharmacology , Humans , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Phosphorylation/genetics , Platelet-Derived Growth Factor/genetics , Platelet-Derived Growth Factor/metabolism , Proto-Oncogene Proteins c-sis , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Retinoblastoma Protein/genetics , Sp1 Transcription Factor/genetics , Sp1 Transcription Factor/metabolism , Up-Regulation/drug effects , Up-Regulation/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...