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

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.

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

Blood ◽

10.1182/blood.v83.4.1086.bloodjournal8341086 ◽

1994 ◽

Vol 83 (4) ◽

pp. 1086-1092 ◽

Cited By ~ 1

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.

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Monoclonal Antibodies Generated against Glycoconjugates Recognize Chemical Linkers

Antibodies ◽

10.3390/antib9030048 ◽

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.

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Total cell content of CR3 (CD11b/CD18) and LFA-1 (CD11a/CD18) in neonatal neutrophils: relationship to gestational age

Blood ◽

10.1182/blood.v87.9.3929.bloodjournal8793929 ◽

1996 ◽

Vol 87 (9) ◽

pp. 3929-3933 ◽

Cited By ~ 47

Author(s):

LT McEvoy ◽

H Zakem-Cloud ◽

MF Tosi

Keyword(s):

Premature Infants ◽

Gestational Age ◽

Rank Correlation ◽

Surface Expression ◽

Total Cell ◽

Preterm Neonates ◽

Enzyme Linked Immunosorbent Assay ◽

Cell Content ◽

Term Neonates ◽

Adult Control

Neonatal neutrophils (PMN) exhibit a well-documented defect in chemotaxis that is associated with several abnormalities of PMN structure and function, including deficient surface expression of CR3 (CD11b/CD18), a critical adhesion molecule, on chemoattractant- activated PMN. We recently documented that deficient surface expression of CR3 on stimulated neonatal PMN is due principally to a deficiency in total cell content of CR3. In the current studies, we tested the hypothesis that total cell CR3 content of PMN is even more profoundly deficient in premature infants and that PMN CR3 content is directly related to gestational age. A sandwich enzyme-linked immunosorbent assay for CR3 showed that PMN lysates from term neonates ( > or = 37 weeks gestation) contain about 60% of adult PMN levels of CR3, whereas PMN from premature infants (range of 27 to 36 weeks gestation) contained a mean of about 30%, ranging from 10% to 48% (P < .001 for term [n = 6] v premature [n = 11] by unpaired t-test). When the relationship between total cell CR3 and gestational age (n = 15) was analyzed, the correlation coefficient was .94 by linear regression, and the Spearman rank correlation was significant with P < .001. PMN content of LFA-1 (CD11a/CD18) was similarly measured for 14 neonates. Term neonates were equivalent to adults in LFA-1 content of their PMN (99.4% +/- 3.2% of paired adult values, n = 6), whereas prematures (28 to 36 weeks gestation) were deficient, overall (69.1% +/- 10.4%, n = 8, P = .035). Below 35 weeks gestation, LFA-1 values ranged from 26% to 65% of paired adult control values, but no infant of > or = 35 weeks gestation had PMN LFA-1 content that was less than 85% of its adult control. We concluded that CR3 total cell content is more profoundly deficient in premature than in term neonates, that at birth there is a direct relationship between PMN CR3 content and gestational age, and that LFA-1 is deficient only in prematures less than 35 weeks of gestational age. Below 30 weeks gestation, CR3 content of PMN approached that seen in genetic deficiency of the CD18 family of leukocyte integrins, or type 1 leukocyte adhesion deficiency, underscoring the severity of this host impairment in very early preterm neonates.

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A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

International Journal of Molecular Sciences ◽

10.3390/ijms22042141 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2141

Author(s):

Srinu Tumpara ◽

Elena Korenbaum ◽

Mark Kühnel ◽

Danny Jonigk ◽

Beata Olejnicka ◽

...

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Western Blotting ◽

Complex Mixture ◽

Immunoglobulin M ◽

Confocal Laser ◽

Enzyme Linked Immunosorbent Assay ◽

Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

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Reevaluating Patient Eligibility for Inotuzumab Ozogamicin Based on CD22 Expression: Is Dim Expression Sufficient?

Current Oncology ◽

10.3390/curroncol28010027 ◽

2020 ◽

Vol 28 (1) ◽

pp. 252-259

Author(s):

Warren Fingrut ◽

Wendy Davis ◽

Eric McGinnis ◽

Karen Dallas ◽

Khaled Ramadan ◽

...

Keyword(s):

Quality Of Life ◽

Monoclonal Antibody ◽

Flow Cytometry ◽

Acute Lymphoblastic Leukemia ◽

Survival Benefit ◽

Lymphoblastic Leukemia ◽

Surface Expression ◽

Inotuzumab Ozogamicin ◽

Cell Acute Lymphoblastic Leukemia

Salvage options for patients with relapsed B-cell acute lymphoblastic leukemia (B-ALL) include inotuzumab ozogamicin (InO), a recombinant, humanized anti-CD22 monoclonal antibody conjugated to the cytotoxic antibiotic calicheamicin. However, the benefit of InO in patients with dim CD22 expression remains unclear. We present a case of a patient with B-ALL who responded to InO despite only dim surface expression of CD22 by flow cytometry, achieving a survival benefit concordant with that reported in the literature and maintaining a good quality of life as a transfusion-independent outpatient. Our observation has broad relevance to clinicians who manage patients with B-ALL who are candidates for InO.

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Multiplatform Physiologic and Metabolic Phenotyping Reveals Microbial Toxicity

mSystems ◽

10.1128/msystems.00123-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 2

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.

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

Journal of Clinical Medicine ◽

10.3390/jcm8101617 ◽

2019 ◽

Vol 8 (10) ◽

pp. 1617 ◽

Cited By ~ 2

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.

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CD80 Expression Correlates with IL-6 Production in THP-1-Like Macrophages Costimulated with LPS and Dialyzable Leukocyte Extract (Transferon®)

Journal of Immunology Research ◽

10.1155/2019/2198508 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Alexis P. Jiménez-Uribe ◽

Hugo Valencia-Martínez ◽

Gregorio Carballo-Uicab ◽

Luis Vallejo-Castillo ◽

Emilio Medina-Rivero ◽

...

Keyword(s):

Molecular Weight ◽

Flow Cytometry ◽

Viral Infections ◽

Surface Expression ◽

Low Molecular Weight ◽

Activated Macrophages ◽

Therapeutic Properties ◽

And Function ◽

Complex Drug ◽

Stimulation Of

Transferon® is a complex drug based on a mixture of low molecular weight peptides. This biotherapeutic is employed as a coadjuvant in clinical trials of several diseases, including viral infections and allergies. Given that macrophages play key roles in pathogen recognition, phagocytosis, processing, and antigen presentation, we evaluated the effect of Transferon® on phenotype and function of macrophage-like cells derived from THP-1 monocytes. We determined the surface expression of CD80 and CD86 by flow cytometry and IL-1β, TNF-α, and IL-6 levels by ELISA. Transferon® alone did not alter the steady state of PMA-differentiated macrophage-like THP-1 cells. On the contrary, simultaneous stimulation of cells with Transferon® and LPS elicited a significant increase in CD80 (P≤0.001) and CD86 (P≤0.001) expression, as well as in IL-6 production (P≤0.05) compared to the LPS control. CD80 expression and IL-6 production exhibited a positive correlation (r=0.6, P≤0.05) in cells exposed to Transferon® and LPS. Our results suggest that the administration of Transferon® induces the expression of costimulatory molecules and the secretion of cytokines in LPS-activated macrophages. Further studies are necessary to determine the implication of these findings in the therapeutic properties of Transferon®.

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