Evaluation of a Whole-Blood Cytokine Release Assay for Use in Measuring Endotoxin Activity of Group B Neisseria meningitidis Vaccines Made from Lipid A Acylation Mutants

ABSTRACT Bacterial endotoxin interacts with the human immune system via complex immunological pathways. The evaluation of endotoxicity is important in the development of safe vaccines and immunomodulatory therapeutics. The Limulus amebocyte lysate (LAL) assay is generally accepted by the FDA for use for the quantification of lipopolysaccharide (LPS), while the rabbit pyrogen test (RPT) is used to estimate pyrogenicity during early development and production. Other in vitro assays, such as cytokine release assays with human whole blood (WB) or peripheral blood mononuclear cells (PBMCs), have also been used and may better estimate the human immunological response to products containing novel LPS molecules. In this study, WB and PBMC interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) release assays were used to estimate the endotoxic activities of purified LPS and native outer membrane vesicle (NOMV) vaccines derived from wild-type (hexa-acylated lipid A) and genetically detoxified (penta- and tetra-acylated lipid A) group B Neisseria meningitidis. A method for quantification of the differences in endotoxicity observed in the WB and PBMC assays is elucidated. The LAL assay was shown to be relatively insensitive to lipid A variations, and the RPT was less sensitive than the cytokine release assay with WB. The IL-6 and TNF-α assays with WB but not the assays with PBMCs distinguished between vaccines containing LPS from penta- and tetra-acylated strains. The high degree of sensitivity of the WB system to LPS variations and the presumed relevance of the use of human tissues to predict toxicity in humans suggest that this assay may be particularly well suited for the safety evaluation of vaccines and therapeutics containing acylation variants of LPS.

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FC 099DECLINING PERITONEAL HOST DEFENCES REVEALED BY EX-VIVO CYTOKINE RELEASE ASSAY OF PERITONEAL DIALYSIS EFFLUENT CELLS

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab141.002 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Rebecca Herzog ◽

Lisa Daniel-Fischer ◽

Isabel Sobieszek ◽

Christoph Aufricht ◽

Klaus Kratochwill

Keyword(s):

Ex Vivo ◽

Significant Proportion ◽

Host Defence ◽

Clinical Staging ◽

Continuous Exposure ◽

Immune Competence ◽

Cytokine Release ◽

Acute Peritonitis ◽

Tnf Α ◽

Release Assay

Abstract Background and Aims Infectious complications occur in a significant proportion of PD patients, limiting long-term applicability. Reduced peritoneal immune-competence, caused by the continuous exposure to PD-fluids, has been described as a therapy-related pathomechanisms, prompting the need for a tool to assess the functional peritoneal immune status. We established an ex-vivo stimulation assay to test host defence mechanisms in only 9ml of PD-effluent. The aim of this study was to analyse basal inflammation and immune-competence in the general PD population at routine conditions to evaluate the assay as surrogate parameter of immune competence and linking it to PD vintage and clinical outcome parameters. Method 147 of 284 (51.8%) adult and paediatric PD patients treated between April 2013 and September 2020 at the local Department of Nephrology were included in the analysis. The study was approved by the local ethics committee and was conducted in accordance with the Declaration of Helsinki. Patients were exclusively treated with neutral pH/multi-chamber PD fluids during the glucose dwells. The majority of the 558 included PD-effluent samples were obtained during standard 4-hours peritoneal equilibration tests (PET) with 3.86% glucose containing PDF. Samples from the pre-PET dwell and at PET time points 1-hour and 4-hours were collected and immediately processed. Additional effluent samples were obtained during unscheduled hospitalization and in the event of an acute peritonitis. Effluent samples were collected directly from the drainage bags into standard 9 ml additive-free sample tubes. For ex-vivo stimulation, 100 ng/ml toll-like receptor (TLR) 4 agonist LPS and TLR2 agonist Pam3Cys were added to the effluent in the 9 ml collection tubes in duplicates and incubated at 37°C for 24h. Unstimulated samples kept in parallel were used as controls. IL-6 and TNF-α concentrations were measured with ELISA in the supernatants. Results Ex-vivo stimulation of peritoneal cells significantly increased the IL-6 and TNF-α release compared to unstimulated controls and resulted in a dwell-time dependent increase, with a significant lower cytokine released at the 1h PET time point. To assess local inflammation IL-6 levels of crude effluent were determined. IL-6 concentrations remained stable over time on PD. Interestingly, we were able to show higher IL-6 levels in CAPD patients in comparison to APD. As chronic exposure to PD-fluids has been shown to dampen the peritoneal immune competence, consecutive peritoneal effluent bags, obtained from patients were analysed. In this subcohort of 183 4h-PET effluents we found a decline in cytokine secretion with time on PD (IL-6 r=-0.27, p=0.00015, TNFa r=-0.25, p=0.00071). In a subgroup the ex-vivo cytokine release of effluent samples from patients with an acute peritonitis was assessed. IL-6 levels of acute peritonitis effluent samples did not differ from the stimulated IL-6 levels of effluent samples without acute peritonitis (2.45 pg/mL vs 2.31 pg/mL, p=0.85, t-test) suggesting that the assay seemingly represents the in-vivo host-defence cytokine release accurately. Conclusion The study provides evidence of a correlation of declining local host defence and duration of PD-therapy. It supports the hypothesis of PD duration-dependent progressive impairment of the ability of the peritoneal immune cells to secrete cytokines in response to a pathogenic stimulus and thereby dampening the global peritoneal immuno-competence. This suggests the utility of this clinically feasible ex-vivo induced cytokine-release assay in peritoneal effluent as a surrogate of the functional peritoneal immune competence. Future analyses need to evaluate the assay as a tool to predict common clinical outcomes and define reference values to facilitate stratification of patient populations, clinical staging and to guide novel therapeutic interventions.

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Structure of the sugar-phosphate moiety of lipid A from lipooligosaccharide of Neisseria meningitidis group B, strain BC5S No. 125

Archives of Microbiology ◽

10.1007/bf00245280 ◽

1992 ◽

Vol 157 (2) ◽

pp. 131-134 ◽

Cited By ~ 6

Author(s):

Vyacheslav L. L'vov ◽

Irina K. Verner ◽

Larisa Yu. Musina ◽

Alexander V. Rodionov ◽

Anatoly V. Ignatenko ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Lipid A ◽

Sugar Phosphate ◽

Phosphate Moiety ◽

Group B

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Deactivation of the lipopolysaccharide antagonist eritoran (E5564) by high-density lipoprotein-associated apolipoproteins

Innate Immunity ◽

10.1177/1753425910394395 ◽

2011 ◽

Vol 18 (1) ◽

pp. 171-178 ◽

Cited By ~ 6

Author(s):

Jacklyn G Fleischer ◽

Dan Rossignol ◽

Gordon A Francis ◽

Teddy Chan ◽

Melvyn Lynn ◽

...

Keyword(s):

Whole Blood ◽

Lipid A ◽

Density Lipoprotein ◽

Antagonistic Activity ◽

High Density ◽

High Density Lipoproteins ◽

Amyloid A ◽

Tnf Α ◽

Signalling Cascade ◽

Pro Inflammatory Cytokines

Lipid A, the active moiety of LPS, exerts its effects through interaction with TLR4, triggering a signalling cascade that results in the release of pro-inflammatory cytokines. Eritoran is a lipid A analogue that competes with LPS for binding to TLR4; however, after intravenous administration, it undergoes a time-dependent deactivation as a consequence of binding to high-density lipoproteins (HDLs). The site of eritoran association with HDL remains unknown. Therefore the aim of this study was to determine if HDL-associated apolipoproteins A1, A2, serum amyloid A (SAA) and C1, inhibit the ability of eritoran to block LPS-induced TNF-α release from whole blood. Eritoran activity after LPS stimulation in human whole blood was assessed in the presence of reconstituted HDL (rHDL) containing different apos. In rHDL, the major apolipoproteins in both the healthy and septic state, A1 and SAA, caused a significant reduction in eritoran antagonistic activity and had a greater effect than minor apolipoproteins A2 and C1. Apolipoproteins associated with HDL are likely to facilitate eritoran deactivation. Apolipoproteins A1 and SAA should be of particular focus as they are the major apos found on HDL in both the healthy and septic state. Further evaluation of the physical association between apolipoproteins and eritoran should be explored.

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Intracellular and Extracellular Cytokine Production by Human Mixed Mononuclear Cells in Response to Group B Streptococci

Infection and Immunity ◽

10.1128/iai.68.1.320-327.2000 ◽

2000 ◽

Vol 68 (1) ◽

pp. 320-327 ◽

Cited By ~ 35

Author(s):

Daniel J. Kwak ◽

Nancy H. Augustine ◽

Wellington G. Borges ◽

Joanna L. Joyner ◽

Wayne F. Green ◽

...

Keyword(s):

Mononuclear Cells ◽

Brefeldin A ◽

Severe Infection ◽

Human Monocyte ◽

Group B Streptococci ◽

Necrosis Factor Alpha ◽

Intracellular Cytokines ◽

Tnf Α ◽

Ifn Γ ◽

Group B

ABSTRACT Group B streptococci (GBS) are a major cause of severe infection in newborns, pregnant females, and other immunocompromised hosts. Infection often includes septicemia, shock, pneumonia, and respiratory failure. In previous studies, we have reported that GBS induce marked production of tumor necrosis factor alpha (TNF-α) by human mononuclear cells. The present study was designed to measure the production of TNF-α as well as additional cytokines, including interleukin 1β (IL-1β), IL-6, IL-8, IL-12, and gamma interferon (IFN-γ) but also to determine from what cells and at what time point during incubation with GBS that these cytokines are produced. Mixed mononuclear cells were incubated with heat-killed GBS, media alone, or 1 μg of Escherichia coli lipopolysaccharide (LPS). Brefeldin A was added to each sample prior to staining, which prevented the export of cytokines by the Golgi apparatus. The cells were then stained with the appropriate conjugated antibodies and analyzed by using a flow cytometer. Results indicate that intracellular cytokines appear, in almost all cases, simultaneous to or before secreted proteins are detected. In contrast to the response to LPS, where TNF-α, IL-1β, IL-6, and IL-8 appear almost simultaneously, the human monocyte response to GBS results in the production of TNF-α but delayed appearance of IL-1β, IL-6, and IL-8. The lymphocyte response to GBS was also strikingly different from that to LPS in that both secreted IFN-γ and IL-12 was detected, while LPS failed to induce production of these critical cytokines. This suggests an important role for TNF-α, IFN-γ, and IL-12 in GBS pathogenesis and/or immunity.

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CpG oligonucleotides increase HBV-specific cytokine responses in whole blood and enhance cytokine release assay sensitivity

Journal of Virological Methods ◽

10.1016/j.jviromet.2017.07.011 ◽

2017 ◽

Vol 248 ◽

pp. 195-201 ◽

Cited By ~ 3

Author(s):

Werner Dammermann ◽

Julia Dornbrack ◽

Katharina Bröker ◽

Frank Bentzien ◽

Stefan Lüth

Keyword(s):

Whole Blood ◽

Cytokine Release ◽

Assay Sensitivity ◽

Cytokine Responses ◽

Cpg Oligonucleotides ◽

Release Assay

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Downregulation of proinflammatory cytokine release in whole blood from septic patients

Blood ◽

10.1182/blood.v85.5.1341.bloodjournal8551341 ◽

1995 ◽

Vol 85 (5) ◽

pp. 1341-1347 ◽

Cited By ~ 326

Author(s):

W Ertel ◽

JP Kremer ◽

J Kenney ◽

U Steckholzer ◽

D Jarrar ◽

...

Keyword(s):

Severe Sepsis ◽

Mrna Expression ◽

Proinflammatory Cytokines ◽

Whole Blood ◽

Mononuclear Cells ◽

Natural Infection ◽

Tnf Alpha ◽

Control Group ◽

Cytokine Release ◽

Septic Group

Using animal models or healthy volunteers, injection of lipopolysaccharide (LPS) or bacteria causes activation of macrophages with excessive synthesis and secretion of proinflammatory cytokines. Although these models mimic the effects of LPS in the host, they may represent more of an experimental expression of endotoxemia than natural infection itself. Therefore, as an ex vivo model of sepsis, whole blood from 15 patients with severe sepsis and 20 control patients without infection was stimulated with LPS to study the kinetics of mRNA expression and release of proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and IL-6. Stimulation of whole blood with 1 microgram/mL LPS resulted in a maximum increase of cytokine secretion in the control group, while a marked (P < .01) depression of TNF-alpha, IL-1 beta, and IL-6 release was observed in the septic group, which persisted up to 10 days after study enrollment. While IL-1 beta mRNA expression was similar in peripheral blood mononuclear cells (PBMCs) harvested from LPS-stimulated whole blood in septic and control patients, the half-life and consequently the expression of TNF-alpha and IL-6 mRNA were strongly reduced in the septic group. These data indicate a downregulatory mechanism of cytokine release in whole blood from patients with severe sepsis that occurs on different levels. Although excessive secretion of proinflammatory cytokines has been considered deleterious for the host, the reduced capacity of PBMCs in whole blood from septic patients to synthesize and secrete proinflammatory cytokines to an inflammatory stimulus may result in immunodeficiency, because these cytokines in low concentrations are involved in the upregulation of essential cellular and humoral immune functions.

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Oral lactobacillus strains reduce cytotoxicity and cytokine release from peripheral blood mononuclear cells exposed to Aggregatibacter actinomycetemcomitans subtypes in vitro.

10.21203/rs.3.rs-16413/v3 ◽

2020 ◽

Author(s):

Nuntiya Pahumunto ◽

Amina Basic ◽

Anna-Karin Östberg ◽

Rawee Teanpaisan ◽

Gunnar Dahlen

Keyword(s):

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Blood Donors ◽

Mononuclear Cells ◽

Aggregatibacter Actinomycetemcomitans ◽

Cytokine Release ◽

Peripheral Blood Mononuclear ◽

Tnf Α ◽

Blood Mononuclear Cells

Abstract Background: This study evaluated the effect of oral lactobacilli on the cytotoxicity and cytokine release from peripheral blood mononuclear cells (PBMCs) when exposed to Aggregatibacter actinomycetemcomitans subtypes in vitro. The supernatants and cell wall extracts (CWEs) of eight A. actinomycetemcomitans strains, representing different subtypes, and three Lactobacillus strains were used. The PBMCs from six blood donors were exposed to supernatants and CWEs of A. actinomycetemcomitans or Lactobacillus strains alone or combinations and untreated cells as control. The cytotoxicity was determined by trypan blue exclusion method and IL-1β secretion by ELISA. TNF-α, IL-6, and IL-8 secretions were measured using Bioplex Multiplex Immunoassay. Results: Supernatants or CWEs from all bacterial strains showed cytotoxicity and IL-1β secretion and the subtypes of A. actinomycetemcomitans showed generally a significantly higher effect on PBMCs than that of the Lactobacillus strains. Two highly toxic A. actinomycetemcomitans strains (JP2 and JP2-like) induced a higher response than all other strains. When combined, Lactobacillus significantly reduced the toxicity and the IL-1β secretion induced by A. acinomycetemcomitans. The effect varied between the subtypes and the reduction was highest for the JP2 and JP2-like strains. The Lactobacillus paracasei strain SD1 had a higher reducing effect than the other Lactobacillus strains. This strain had a consistent reducing effect on all subtypes of A. actinomycetemcomitans cytotoxicity, and release of IL-1β, IL-6, IL-8, and TNF-α from PBMCs of the blood donors. A strong and significant variation in cytokine release between the six blood donors was noticed.Conclusions: Lactobacillus spp. and L. paracasei SD1 in particular, showed a limited but statistically significant reducing interaction with A. actinomycetemcomitans toxicity and release of cytokines in vitro.

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Functional Activities and Epitope Specificity of Human and Murine Antibodies against the Class 4 Outer Membrane Protein (Rmp) of Neisseria meningitidis

Infection and Immunity ◽

10.1128/iai.67.3.1267-1276.1999 ◽

1999 ◽

Vol 67 (3) ◽

pp. 1267-1276 ◽

Cited By ~ 38

Author(s):

Einar Rosenqvist ◽

Alexis Musacchio ◽

Audun Aase ◽

E. Arne Høiby ◽

Ellen Namork ◽

...

Keyword(s):

Membrane Protein ◽

Neisseria Meningitidis ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Membrane Vesicle ◽

Bactericidal Effect ◽

Outer Membrane Vesicle ◽

Human Sera ◽

High Concentrations ◽

Group B

ABSTRACT Antibodies against the class 4 outer membrane protein (OMP) fromNeisseria meningitidis have been purified from sera from vaccinees immunized with the Norwegian meningococcal group B outer membrane vesicle vaccine. The human sera and purified antibodies reacted strongly with the class 4 OMP in immunoblots, whereas experiments with whole bacteria showed only weak reactions, indicating that the antibodies mainly reacted with parts of the class 4 molecule that were not exposed. The purified human anti-class 4 OMP antibodies and the monoclonal antibodies (MAbs) were neither bactericidal nor opsonic against live meningococci. Three new MAbs against the class 4 OMP were generated and compared with other, previously described MAbs. Three linear epitopes in different regions of the class 4 OMP were identified by the reaction of MAbs with synthetic peptides. The MAbs showed no blocking effect on bactericidal activity of MAbs against other OMPs. However, one of the eight purified human anti-class 4 OMP antibody preparations, selected from immunoblot reactions among sera from 27 vaccinees, inhibited at high concentrations the bactericidal effect of a MAb against the class 1 OMP. However, these antibodies were not vaccine induced, as they were present also before vaccination. Therefore, this study gave no evidence that vaccination with a meningococcal outer membrane vesicle vaccine containing the class 4 OMP induces blocking antibodies. Our data indicated that the structure of class 4 OMP does not correspond to standard β-barrel structures of integral OMPs and that no substantial portion of the OmpA-like C-terminal region of this protein is located at the surface of the outer membrane.

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TLR agonist combinations that stimulate Th type I polarizing responses from human neonates

Innate Immunity ◽

10.1177/1753425918771178 ◽

2018 ◽

Vol 24 (4) ◽

pp. 240-251 ◽

Cited By ~ 11

Author(s):

Naveen Surendran ◽

Andrea Simmons ◽

Michael E Pichichero

Keyword(s):

Cord Blood ◽

Lipid A ◽

Mononuclear Cells ◽

Poly I:C ◽

Type I ◽

Monophosphoryl Lipid A ◽

Tnf Α ◽

Tlr Agonist ◽

Ifn Γ ◽

Childhood Vaccines

Each year millions of neonates die due to vaccine preventable infectious diseases. Our study seeks to develop novel neonatal vaccines and improve immunogenicity of early childhood vaccines by incorporating TLR agonist-adjuvant combinations that overcome the inherent neonatal Th2 bias and stimulate Th1 polarizing response from neonatal APCs. We systematically stimulated cord blood mononuclear cells with single and multiple combinations of TLR agonists and measured levels of IL-12p70, IFN-γ, IFN-α, IL-10, IL-13, TNF-α, IL-6 and IL-1β from cell culture supernatants. APC-specific surface expression levels of costimulatory markers CD40, CD83 and PD-L1 were assessed by flow cytometry. Whole blood assays were included to account for the effect of plasma inhibitory factors and APC intracellular TNF-α and IL-12p40 secretions were measured. We found robust Th1 polarizing IL-12p70, IFN-γ and IFN-α responses when cord blood APCs were stimulated with TLR agonist combinations that contained Poly I:C, Monophosphoryl Lipid A (MPLA) or R848. Addition of class A CpG oligonucleotide (ODN) to Th1 polarizing TLR agonist combinations significantly reduced cord blood IL-12p70 and IFN-γ levels and addition of a TLR2 agonist induced significantly high Th2 polarizing IL-13. Multi-TLR agonist combinations that included R848 induced lower inhibitory PD-L1 expression on cord blood classical dendritic cells than CpG ODN-containing combinations. Incorporation of combination adjuvants containing TLR3, TLR4 and TLR7/8 agonists to neonatal vaccines may be an effective strategy to overcome neonatal Th2 bias.

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Cellular response of blood-borne immune cells to PEEU fiber meshes

Clinical Hemorheology and Microcirculation ◽

10.3233/ch-219114 ◽

2021 ◽

Vol 79 (1) ◽

pp. 205-216

Author(s):

Janita A. Maring ◽

Matthias Becker ◽

Wing Tai Tung ◽

Christof Stamm ◽

Nan Ma ◽

...

Keyword(s):

Immune Response ◽

Immune Cells ◽

Peripheral Blood ◽

Cellular Response ◽

Mononuclear Cells ◽

Macrophage Polarization ◽

T Cell Proliferation ◽

Cytokine Release ◽

Tnf Α ◽

Activated Monocytes

BACKGROUND: Polymeric materials have been widely used as artificial grafts in cardiovascular applications. These polymeric implants can elicit a detrimental innate and adaptive immune response after interacting with peripheral blood. A surface modification with components from extracellular matrices (ECM) may minimize the activation of immune cells from peripheral blood. The aim of this study is to compare the cellular response of blood-born immune cells to the fiber meshes from polyesteretherurethane (PEEUm) and PEEUm with ECM coating (PEEUm + E). MATERIALS AND METHODS: Electrospun PEEUm were used as-is or coated with human cardiac ECM. Different immune cells were isolated form human peripheral blood. Cytokine release profile from naïve and activated monocytes was assessed. Macrophage polarization and T cell proliferation, as indication of immune response were evaluated. RESULTS: There was no increase in cytokine release (IL-6, TNF-α, and IL-10) from activated monocytes, macrophages and mononuclear cells on PEEUm; neither upon culturing on PEEUm + E. Naïve monocytes showed increased levels of IL-6 and TNF-α, which were not present on PEEUm + E. There was no difference on monocyte derived macrophage polarization towards pro-inflammatory M1 or anti-inflammatory M2 on PEEUm and PEEUm + E. Moreover, T cell proliferation was not increased upon interacting with PEEUm directly. CONCLUSION: As PEEUm only elicits a minimal response from naïve monocytes but not from monocytes, peripheral blood mononuclear cells (PBMCs) or T cells, the slight improvement in response to PEEUm + E might not justify the additional effort of coating with a human ECM.

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