scholarly journals Monoclonal Antibodies Generated against Glycoconjugates Recognize Chemical Linkers

Antibodies ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 48
Author(s):  
Jessica Ramadhin ◽  
Vanessa Silva-Moraes ◽  
Thomas Norberg ◽  
Donald Harn
Keyword(s):  
Drug Delivery ◽  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Structure And Function ◽  
Enzyme Linked Immunosorbent Assay ◽  
Incomplete Freund’S Adjuvant ◽  
Delivery Platform ◽  
Igg1 Mabs ◽  
Human Milk Oligosaccharide ◽  
And Function

Monoclonal antibodies (mAbs) that recognize glycans are useful tools to assess carbohydrates’ structure and function. We sought to produce IgG mAbs to the human milk oligosaccharide (HMO), lacto-N-fucopentaose III (LNFPIII). LNFPIII contains the Lewisx antigen, which is found on the surface of schistosome parasites. mAbs binding the Lewisx antigen are well-reported in the literature, but mAbs recognizing HMO structures are rare. To generate mAbs, mice were immunized with LNFPIII-DEX (P3DEX) plus CpGs in VacSIM®, a novel vaccine/drug delivery platform. Mice were boosted with LNFPIII-HSA (P3HSA) plus CpGs in Incomplete Freund’s Adjuvant (IFA). Splenocytes from immunized mice were used to generate hybridomas and were screened against LNFPIII conjugates via enzyme-linked immunosorbent assay (ELISA). Three positive hybridomas were expanded, and one hybridoma, producing IgG and IgM antibodies, was cloned via flow cytometry. Clone F1P2H4D8D5 was selected because it produced IgG1 mAbs, but rescreening unexpectedly showed binding to both LNFPIII and lacto-N-neotetraose (LNnT) conjugates. To further assess the specificity of the mAb, we screened it on two glycan microarrays and found no significant binding. This finding suggests that the mAb binds to the acetylphenylenediamine (APD) linker-spacer structure of the conjugate. We present the results herein, suggesting that our new mAb could be a useful probe for conjugates using similar linker spacer structures.

scholarly journals Deficient total cell content of CR3 (CD11b) in neonatal neutrophils

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1086-1092 ◽  
Author(s):  
N Abughali ◽  
M Berger ◽  
MF Tosi
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Surface Expression ◽  
Structure And Function ◽  
Total Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Beta Chain ◽  
Cell Content ◽  
Signaling Mechanisms ◽  
And Function

Abstract Neonatal neutrophils (PMN) show a well-documented defect in chemotaxis that is associated with several abnormalities of PMN structure and function, including deficient surface expression of CR3 (CD11b), a critical adhesion molecule, on chemoattractant-activated PMN. After activation of PMN with additional stimuli including calcium ionophores, we also found deficient surface CR3 (but normal CR1) expression on neonatal PMN suggesting that abnormal signaling mechanisms are not likely to explain the deficient CR3 expression on activated neonatal PMN. Therefore, we hypothesized that deficient surface expression of CR3 on stimulated neonatal neutrophils is caused by a deficiency in total cell content of CR3. We tested this hypothesis using three different methods to compare the total quantity of CR3 in neonatal versus adult PMN. Western blotting of serial twofold dilutions of PMN lysates from five adult and neonatal pairs, using a monoclonal antibody (MoAb) against CR3 (21PM19C), consistently showed diminished CR3 content in neonatal PMN. A sandwich enzyme-linked immunosorbent assay, in which the CR3 heterodimers in PMN lysates were captured by MoAb to the beta-chain, CD18 (R15.7), then detected with a biotinylated MoAb to the alpha-chain, CD11b (anti-Mac-1), showed that neonatal PMN lysates contain about 66% of adult PMN levels of CR3 (P < 0.03; n = 6). PMN fixed with paraformaldehyde and permeabilized with saponin were studied by immunofluorescence flow cytometry to determine total (surface plus intracellular) CR3 content using phycoerythrin-conjugated MoAb to CR3 (anti-Leu15). Mean total cell CR3 content (in relative fluorescence units) was 58 +/- 14 for adult PMN and 27 +/- 6 for neonatal PMN (n=5; P=0.013). In each method, total cell content of CR1 was equivalent for neonatal versus adult PMN. We conclude that neonatal PMN are markedly deficient in total cell CR3 content compared with adult PMN. This result provides a primary explanation for deficient CR3 surface expression on activated neonatal PMN that, in turn, may be important in the chemotactic defect of these cells.

scholarly journals Deficient total cell content of CR3 (CD11b) in neonatal neutrophils

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1086-1092 ◽  
Author(s):  
N Abughali ◽  
M Berger ◽  
MF Tosi
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Surface Expression ◽  
Structure And Function ◽  
Total Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Beta Chain ◽  
Cell Content ◽  
Signaling Mechanisms ◽  
And Function

Neonatal neutrophils (PMN) show a well-documented defect in chemotaxis that is associated with several abnormalities of PMN structure and function, including deficient surface expression of CR3 (CD11b), a critical adhesion molecule, on chemoattractant-activated PMN. After activation of PMN with additional stimuli including calcium ionophores, we also found deficient surface CR3 (but normal CR1) expression on neonatal PMN suggesting that abnormal signaling mechanisms are not likely to explain the deficient CR3 expression on activated neonatal PMN. Therefore, we hypothesized that deficient surface expression of CR3 on stimulated neonatal neutrophils is caused by a deficiency in total cell content of CR3. We tested this hypothesis using three different methods to compare the total quantity of CR3 in neonatal versus adult PMN. Western blotting of serial twofold dilutions of PMN lysates from five adult and neonatal pairs, using a monoclonal antibody (MoAb) against CR3 (21PM19C), consistently showed diminished CR3 content in neonatal PMN. A sandwich enzyme-linked immunosorbent assay, in which the CR3 heterodimers in PMN lysates were captured by MoAb to the beta-chain, CD18 (R15.7), then detected with a biotinylated MoAb to the alpha-chain, CD11b (anti-Mac-1), showed that neonatal PMN lysates contain about 66% of adult PMN levels of CR3 (P < 0.03; n = 6). PMN fixed with paraformaldehyde and permeabilized with saponin were studied by immunofluorescence flow cytometry to determine total (surface plus intracellular) CR3 content using phycoerythrin-conjugated MoAb to CR3 (anti-Leu15). Mean total cell CR3 content (in relative fluorescence units) was 58 +/- 14 for adult PMN and 27 +/- 6 for neonatal PMN (n=5; P=0.013). In each method, total cell content of CR1 was equivalent for neonatal versus adult PMN. We conclude that neonatal PMN are markedly deficient in total cell CR3 content compared with adult PMN. This result provides a primary explanation for deficient CR3 surface expression on activated neonatal PMN that, in turn, may be important in the chemotactic defect of these cells.

scholarly journals Multiplatform Physiologic and Metabolic Phenotyping Reveals Microbial Toxicity

mSystems ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Jingwei Cai ◽  
Robert G. Nichols ◽  
Imhoi Koo ◽  
Zachary A. Kalikow ◽  
Limin Zhang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Flow Cytometry ◽  
Free Radical ◽  
Gut Microbiota ◽  
Structure And Function ◽  
Metabolic Phenotyping ◽  
And Function

ABSTRACTThe gut microbiota is susceptible to modulation by environmental stimuli and therefore can serve as a biological sensor. Recent evidence suggests that xenobiotics can disrupt the interaction between the microbiota and host. Here, we describe an approach that combinesin vitromicrobial incubation (isolated cecal contents from mice), flow cytometry, and mass spectrometry- and1H nuclear magnetic resonance (NMR)-based metabolomics to evaluate xenobiotic-induced microbial toxicity. Tempol, a stabilized free radical scavenger known to remodel the microbial community structure and functionin vivo, was studied to assess its direct effect on the gut microbiota. The microbiota was isolated from mouse cecum and was exposed to tempol for 4 h under strict anaerobic conditions. The flow cytometry data suggested that short-term tempol exposure to the microbiota is associated with disrupted membrane physiology as well as compromised metabolic activity. Mass spectrometry and NMR metabolomics revealed that tempol exposure significantly disrupted microbial metabolic activity, specifically indicated by changes in short-chain fatty acids, branched-chain amino acids, amino acids, nucleotides, glucose, and oligosaccharides. In addition, a mouse study with tempol (5 days gavage) showed similar microbial physiologic and metabolic changes, indicating that thein vitroapproach reflectedin vivoconditions. Our results, through evaluation of microbial viability, physiology, and metabolism and a comparison ofin vitroandin vivoexposures with tempol, suggest that physiologic and metabolic phenotyping can provide unique insight into gut microbiota toxicity.IMPORTANCEThe gut microbiota is modulated physiologically, compositionally, and metabolically by xenobiotics, potentially causing metabolic consequences to the host. We recently reported that tempol, a stabilized free radical nitroxide, can exert beneficial effects on the host through modulation of the microbiome community structure and function. Here, we investigated a multiplatform phenotyping approach that combines high-throughput global metabolomics with flow cytometry to evaluate the direct effect of tempol on the microbiota. This approach may be useful in deciphering how other xenobiotics directly influence the microbiota.

scholarly journals Sub-Inhibitory Clindamycin and Azithromycin reduce S. aureus Exoprotein Induced Toxicity, Inflammation, Barrier Disruption and Invasion

2019 ◽  
Vol 8 (10) ◽  
pp. 1617 ◽  
Author(s):  
Hu ◽  
Ramezanpour ◽  
Hayes ◽  
Liu ◽  
Psaltis ◽  
...  
Keyword(s):  
Structure And Function ◽  
Mucosal Barrier ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Synthesis Inhibitor ◽  
Barrier Structure ◽  
Synthesis Inhibitor ◽  
Barrier Disruption ◽  
Human Nasal Epithelial Cells ◽  
Invasive Capacity ◽  
And Function

Background: Chronic rhinosinusitis (CRS) is defined as a chronic inflammation of the nose and paranasal sinus mucosa associated with relapsing infections—particularly with S. aureus. Long-term treatments with protein synthesis inhibitor antibiotics have been proposed to reduce inflammation in the context chronic severe inflammatory airway pathologies, including CRS. This study assessed the effect of subinhibitory clindamycin and azithromycin on S. aureus exoprotein induced inflammation, toxicity and invasiveness. Methods: S. aureus ATCC51650 and two clinical isolates grown in planktonic and biofilm form were treated with subinhibitory clindamycin and azithromycin. Exoproteins were collected and applied to primary human nasal epithelial cells (HNECs) in monolayers and at air-liquid interface. This was followed by lactate dehydrogenase (LDH), enzyme-linked immunosorbent assay (ELISA), Transepithelial Electrical Resistance (TEER) and paracellular permeability assays to assess the effect on cell toxicity, inflammatory cytokine production and mucosal barrier structure and function, respectively. The effect of these treatments was tested as well on the S. aureus invasiveness of HNECs. Results: Subinhibitory clindamycin reduced S. aureus exoprotein production in planktonic and biofilm form, thereby blocking exoprotein-induced toxicity, reversing its detrimental effects on mucosal barrier structure and function and modulating its inflammatory properties. Sub-inhibitory azithromycin had similar effects—albeit to a lesser extent. Furthermore, clindamycin—but not azithromycin—treated S. aureus lost its invasive capacity of HNECs. Conclusion: Subinhibitory clindamycin and azithromycin reduce S. aureus exoprotein production, thereby modulating the inflammatory cascade by reducing exoprotein-induced toxicity, inflammation, mucosal barrier disruption and invasiveness.

Beyond Fmoc: a review of aromatic peptide capping groups

2020 ◽  
Vol 8 (5) ◽  
pp. 863-877 ◽  
Author(s):  
Adam D. Martin ◽  
Pall Thordarson
Keyword(s):  
Drug Delivery ◽  
Tissue Engineering ◽  
Structure And Function ◽  
Short Peptides ◽  
Peptide Structure ◽  
Energy Materials ◽  
Self Assembling ◽  
And Function

Self-assembling short peptides have widespread applications in energy materials, tissue engineering, sensing and drug delivery. In this review we discuss the effect of functional N-terminal capping groups on peptide structure and function.

scholarly journals High-density lipoproteins for therapeutic delivery systems

2016 ◽  
Vol 4 (2) ◽  
pp. 188-197 ◽  
Author(s):  
R. Kannan Mutharasan ◽  
Linda Foit ◽  
C. Shad Thaxton
Keyword(s):  
Drug Delivery ◽  
Delivery Systems ◽  
Structure And Function ◽  
High Density ◽  
High Density Lipoproteins ◽  
Delivery Vehicle ◽  
Drug Delivery Vehicle ◽  
Therapeutic Delivery ◽  
And Function

High-density lipoproteins are a class of natural nanostructures with multiple desirable properties to model in a drug delivery vehicle. Here we review the structure and function of high-density lipoproteins, and their use as therapeutic delivery systems.

scholarly journals Controlling Structure and Function of Polymeric Drug Delivery Nanoparticles Using Microfluidics

2017 ◽  
Vol 14 (8) ◽  
pp. 2595-2606 ◽  
Author(s):  
Aman Bains ◽  
Yimeng Cao ◽  
Sundiata Kly ◽  
Jeremy E. Wulff ◽  
Matthew G. Moffitt
Keyword(s):  
Drug Delivery ◽  
Structure And Function ◽  
Polymeric Drug Delivery ◽  
Polymeric Drug ◽  
And Function

scholarly journals Pulmonary applications and toxicity of engineered nanoparticles

2008 ◽  
Vol 295 (3) ◽  
pp. L400-L411 ◽  
Author(s):  
Jeffrey W. Card ◽  
Darryl C. Zeldin ◽  
James C. Bonner ◽  
Earle R. Nestmann
Keyword(s):  
Drug Delivery ◽  
Pulmonary Toxicity ◽  
Pulmonary Arteries ◽  
Structure And Function ◽  
Engineered Nanoparticles ◽  
Systemic Delivery ◽  
Imaging Capability ◽  
Potential Safety ◽  
And Function ◽  
Prime Target

Because of their unique physicochemical properties, engineered nanoparticles have the potential to significantly impact respiratory research and medicine by means of improving imaging capability and drug delivery, among other applications. These same properties, however, present potential safety concerns, and there is accumulating evidence to suggest that nanoparticles may exert adverse effects on pulmonary structure and function. The respiratory system is susceptible to injury resulting from inhalation of gases, aerosols, and particles, and also from systemic delivery of drugs, chemicals, and other compounds to the lungs via direct cardiac output to the pulmonary arteries. As such, it is a prime target for the possible toxic effects of engineered nanoparticles. The purpose of this article is to provide an overview of the potential usefulness of nanoparticles and nanotechnology in respiratory research and medicine and to highlight important issues and recent data pertaining to nanoparticle-related pulmonary toxicity.

scholarly journals Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor

1986 ◽  
Vol 235 (1) ◽  
pp. 199-208 ◽  
Author(s):  
M A Soos ◽  
K Siddle ◽  
M D Baron ◽  
J M Heward ◽  
J P Luzio ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Insulin Receptor ◽  
Human Insulin ◽  
Human Placenta ◽  
Structure And Function ◽  
Beta Subunits ◽  
Intact Cells ◽  
Receptor Structure ◽  
Human Insulin Receptor ◽  
And Function

Monoclonal antibodies for the human insulin receptor were produced following immunization of mice with IM-9 lymphocytes and/or purified placental receptor. Four separate fusions yielded 28 antibodies, all of which reacted with receptor from human placenta, liver and IM-9 cells. Some antibodies cross-reacted to varying degrees with receptor from rabbit, cow, pig and sheep, but none reacted with rat receptor. At least 10 distinct epitopes were recognized as indicated by species specificity and binding competition experiments. All of these epitopes appeared to be on extracellular domains of the receptor as shown by binding of antibodies to intact cells. In some cases the epitopes were further localized to alpha or beta subunits by immunoblotting. Several antibodies inhibited binding of 125I-insulin to the receptor, some had no effect on binding, and others enhanced the binding of 125I-insulin. It is concluded that these antibodies will be valuable probes of receptor structure and function.

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