scholarly journals Method for the Selective Measurement of Amino-Terminal Variants of Procalcitonin

2009 ◽  
Vol 55 (9) ◽  
pp. 1672-1679 ◽  
Author(s):  
Joachim Struck ◽  
Martina Strebelow ◽  
Sonja Tietz ◽  
Christine Alonso ◽  
Nils G Morgenthaler ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Bacterial Infections ◽  
Cross Reactivity ◽  
Functional Assay ◽  
Human Endotoxemia ◽  
Amino Terminal ◽  
Endotoxemia Model ◽  
Sandwich Assays ◽  
Kinetics Of

Abstract Background: Procalcitonin (PCT) is an established marker for diagnosing and monitoring bacterial infections. Full-length PCT [116 amino acids that make up procalcitonin (PCT1–116)] can be truncated, leading to des-Ala-Pro-PCT (des-Alanin-Prolin-Procalcitonin; PCT3–116). Current immunoassays for PCT (“total PCT”) use antibodies directed against internal epitopes and are unable to distinguish amino-terminal PCT variants. Here we describe the development of monoclonal antibodies recognizing the amino-termini of PCT1–116 and PCT3–116 and their use in the selective measurement of these PCT species. Methods: With newly developed monoclonal antibodies against the amino-termini of PCT1–116 and PCT3–116, and an antibody against the katacalcin moiety of PCT, we developed and characterized immunoluminometric assays for the 2 PCT peptides. We comparatively assessed the kinetics of PCT variants in a human endotoxemia model. Results: Monoclonal antibodies against the amino-termini of PCT1–116 and PCT3–116 showed <1% cross-reactivity with other PCT-related peptides. The sandwich assays for PCT1–116 and PCT3–116 had functional assay sensitivities of 5 and 1.2 pmol/L, respectively, and exhibited recoveries within 20% of expected values. Plasma PCT1–116 was stable for 6 h at 22 °C and 24 h at 4 °C, and PCT3–116 was stable for at least 24 h at both temperatures. During experimental endotoxemia in healthy people, both PCT1–116 and PCT3–116 increased early in parallel with total PCT, but further increases in PCT1–116 were significantly slower than for PCT3–116 (P = 0.0049) and total PCT (P = 0.0024). Conclusions: The new assays selectively measure PCT1–116 and PCT3–116. Both PCT species increase early during endotoxemia but differ in their kinetics thereafter. The selective measurement of PCT species with different in vivo kinetics may be useful in improving PCT-guided therapies.

Time-course analysis of hepcidin, serum iron, and plasma cytokine levels in humans injected with LPS

Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1864-1866 ◽  
Author(s):  
Erwin Kemna ◽  
Peter Pickkers ◽  
Elizabeta Nemeth ◽  
Hans van der Hoeven ◽  
Dorine Swinkels
Keyword(s):  
Serum Iron ◽  
Time Course ◽  
Regulatory System ◽  
Peptide Hormone ◽  
Time Frame ◽  
Human Endotoxemia ◽  
Plasma Cytokines ◽  
Endotoxemia Model ◽  
Iron Parameters

Abstract Hepatic peptide hormone hepcidin is the key regulator of iron metabolism and the mediator of anemia of inflammation. Previous studies indicated that interleukin-6 (IL-6) mediates hepcidin increase and consequent hypoferremia during inflammation. Here we used an in vivo human endotoxemia model to analyze the effects of lipopolysaccharide (LPS) as a more upstream inflammation activator. The temporal associations between plasma cytokines, hepcidin levels, and serum iron parameters were studied in 10 healthy individuals after LPS injection. IL-6 was dramatically induced within 3 hours after injection, and urinary hepcidin peaked within 6 hours, followed by a significant decrease in serum iron. Serum prohepcidin showed no significant change within a 22-hour time frame. These in vivo human results confirm the importance of the IL-6-hepcidin axis in the development of hypoferremia in inflammation and highlight the rapid responsiveness of this iron regulatory system. (Blood. 2005;106: 1864-1866)

scholarly journals E. coliEndotoxin Modulates the Expression of Sirtuin Proteins in PBMC in Humans

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Angela Storka ◽  
Gerhard Führlinger ◽  
Martin Seper ◽  
Lisa Wang ◽  
Michael Jew ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Mononuclear Cells ◽  
Metabolic Diseases ◽  
Peripheral Blood Mononuclear ◽  
Human Endotoxemia ◽  
Lps Challenge ◽  
Endotoxemia Model ◽  
Regulatory Functions

Background. Sirtuin (SIRT) proteins are class I histone deacetylases displaying gene regulatory functions in inflammatory, cancer, and metabolic diseases. These SIRT actions involve the nuclear factorκB and its inhibitor IκB pathway. However, the regulation of SIRTin vivois still unclear.Material and Methods. In a human endotoxemia model, 20 healthy male subjects received an intravenous bolus of 2 ng/kg body weightEscherichia coliendotoxin (LPS). SIRT expression was investigated in peripheral blood mononuclear cells (PBMC) with qPCR and Western blot before and 3 hours, 6 hours, and 24 hours after LPS challenge. Additionally, SIRT regulation was studiedin vitroin cultivated PBMC after incubation with 20 ng/mL LPS.Results. A downregulation by >40% of SIRT1 mRNA was detectable 3 hours after LPS and of SIRT3 mRNA 6 hours after LPS. SIRT3, IκBα, and IκB-βprotein expressions were decreased 3 and 6 hours after LPS. SIRT2 mRNA or protein expression did not change following LPS. These findings were consistentin vitroand associated with augmented phosphorylation of IκB-β.Discussion. In thisE. coliendotoxemia model, SIRT1 and SIRT3 mRNA expressions in PBMC in humans were reduced after LPS challenge. This suggests that SIRT may represent an inflammatory target proteinin vivo.

scholarly journals Comparison of different lots of endotoxin and evaluation of in vivo potency over time in the experimental human endotoxemia model

2019 ◽  
Vol 25 (1) ◽  
pp. 34-45 ◽  
Author(s):  
Dorien Kiers ◽  
Guus P. Leijte ◽  
Jelle Gerretsen ◽  
Jelle Zwaag ◽  
Matthijs Kox ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Clinical Signs ◽  
Systemic Inflammatory Response ◽  
Bolus Administration ◽  
Continuous Administration ◽  
Human Endotoxemia ◽  
Inflammatory Parameters ◽  
Endotoxemia Model ◽  
Over Time

The experimental human endotoxemia model is used to study the systemic inflammatory response in vivo. The previously used lot of endotoxin, which was used for over a decade, is no longer approved for human use and a new Good Manufacturing Practices-grade batch has become available. We compared the inflammatory response induced by either bolus or continuous administration of either the previously used lot #1188844 or new lots of endotoxin (#94332B1 and #94332B4). Compared with lot #1188844, bolus administration of lot #94332B1 induced a more pronounced systemic inflammatory response including higher plasma levels of pro-inflammatory cytokines and more pronounced clinical signs of inflammation. In contrast, continuous infusion of lot #94332B4 resulted in a slightly less pronounced inflammatory response compared with lot #1188844. Furthermore, we evaluated whether lot #1188844 displayed in vivo potency loss by reviewing inflammatory parameters obtained from 17 endotoxemia studies performed in our centre between 2007 and 2016. Despite inter-study variability in endotoxemia-induced effects on temperature, heart rate, symptoms, and leukocyte counts, the magnitude of these effects did not decrease over time. In conclusion, although all lots of endotoxin induce a pronounced inflammatory response, the magnitude differs between lots. We observed no potency loss of endotoxin over time.

scholarly journals High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones Protect Mice from Lethal Pseudomonas aeruginosa Infections

2013 ◽  
Vol 80 (2) ◽  
pp. 462-469 ◽  
Author(s):  
Soumya Palliyil ◽  
Christina Downham ◽  
Ian Broadbent ◽  
Keith Charlton ◽  
Andrew J. Porter
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Monoclonal Antibodies ◽  
Bacterial Infections ◽  
Recombinant Antibody ◽  
Cell Communication ◽  
High Sensitivity ◽  
Therapeutic Benefit ◽  
Cross Reactivity ◽  
Content Type ◽  
Homoserine Lactones

ABSTRACTA number of bacteria, including pathogens likePseudomonas aeruginosa, utilize homoserine lactones (HSLs) as quorum sensing (QS) signaling compounds and engage in cell-to-cell communication to coordinate their behavior. Blocking this bacterial communication may be an attractive strategy for infection control as QS takes a central role inP. aeruginosabiology. In this study, immunomodulation of HSL molecules by monoclonal antibodies (MAbs) was used as a novel approach to preventP. aeruginosainfections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection. Using sheep immunization and recombinant antibody technology, a panel of sheep-mouse chimeric MAbs were generated which recognized HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. These MAbs retained their nanomolar sensitivity in complex matrices and were able to recognize HSLs inP. aeruginosacultures grown in the presence of urine. In a nematode slow-killing assay, HSL MAbs significantly increased the survival of worms fed on the antibiotic-resistant strain PA058. The therapeutic benefit of these MAbs was further studied using a mouse model ofPseudomonasinfection in which groups of mice treated with HSL-2 and HSL-4 MAbs survived, 7 days after pathogen challenge, in significantly greater numbers (83 and 67%, respectively) compared with the control groups. This body of work has provided early proof-of-concept data to demonstrate the potential of HSL-specific, monoclonal antibodies as theranostic clinical leads suitable for the diagnosis, prevention, and treatment of life-threatening bacterial infections.

In vivo interaction of endotoxin and recombinant bactericidal/permeability-increasing protein (rBPI23): Hemodynamic effects in a human endotoxemia model

2002 ◽  
Vol 140 (4) ◽  
pp. 228-235 ◽  
Author(s):  
Wilbert T. Jellema ◽  
D.Peter Veerman ◽  
Robbert J. de Winter ◽  
Karel H. Wesseling ◽  
Sander J.H. van Deventer ◽  
...  
Keyword(s):  
Hemodynamic Effects ◽  
Human Endotoxemia ◽  
Endotoxemia Model

Sensitive Measurement of Thrombopoietin by a Monoclonal Antibody Based Sandwich Enzyme-linked Immunosorbent Assay

1997 ◽  
Vol 78 (04) ◽  
pp. 1262-1267 ◽  
Author(s):  
Claudia C Folman ◽  
Albert E G K von dem Borne ◽  
Irma H J A M Rensink ◽  
Winald Gerritsen ◽  
C Ellen van der Schoot ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Platelet Transfusion ◽  
Large Scale ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reliable Tool

SummaryIn this report a sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of plasma thrombopoietin (Tpo) is described that is solely based on monoclonal antibodies (MoAbs).The assay has an intra and inter-assay variance of 5-7% and 7-13%, respectively. Native and recombinant human Tpo (rhTpo) were recognized equally well, no cross reactivity with other cytokines was found and rhTpo added to plasma and serum was completely recovered. With the ELISA, Tpo concentrations in EDTA-anticoagulated plasma of all controls (n = 193) could be determined, since the limit of detection (2 ± 0.8 A.U./ml, mean ± sd) was lower than the concentration found in controls (11 ± 8 A.U./ml, mean ± sd; 2.5th-97.5th percentile: 4-32 A.U./ml). Tpo levels in serum were on average 3.4 times higher than in plasma.We showed in vivo that Tpo is bound by platelets, as in thrombocytopenic patients (n = 5) a platelet transfusion immediately led to a drop in plasma Tpo level, whereas in patients receiving chemotherapy the induced thrombocytopenia was followed by a rise in plasma Tpo levels.In summary, these results indicate that this ELISA is a reliable tool for Tpo measurements and is applicable for large scale studies.

The in vivo kinetics of tissue factor messenger RNA expression during human endotoxemia: relationship with activation of coagulation

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 554-559 ◽  
Author(s):  
Rendrik F. Franco ◽  
Evert de Jonge ◽  
Pascale E. P. Dekkers ◽  
Janneke J. Timmerman ◽  
C. Arnold Spek ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Tissue Factor ◽  
Messenger Rna ◽  
Thrombin Generation ◽  
Mrna Levels ◽  
Total Blood ◽  
Human Endotoxemia ◽  
Lps Challenge ◽  
Kinetics Of

Abstract Triggering of the tissue factor (TF)-dependent coagulation pathway is considered to underlie the generation of a procoagulant state during endotoxemia. To determine the in vivo pattern of monocytic TF messenger RNA (mRNA) expression during endotoxemia, 10 healthy volunteers were injected with lipopolysaccharide (LPS, 4 ng/kg) and blood was collected before and 0.5, 1, 2, 3, 4, 6, 8, and 24 hours after LPS administration. Total blood RNA was isolated and amplified by NASBA (nucleic acid sequence-based amplification), followed by quantitation of TF mRNA by an electrochemiluminescence (ECL) assay. To compare the pattern of coagulation activation with the kinetics of monocytic TF mRNA expression, we measured plasma levels of markers of thrombin generation, thrombin-antithrombin (TAT) complexes, and prothrombin fragment 1 + 2 (F1 + 2). Baseline value (mean ± SEM) of the number of TF mRNA molecules per monocytic cell was 0.08 ± 0.02. A progressive and significant (P < .0001) increase in TF expression was observed after LPS injection (+0.5 hour: 0.3 ± 0.1, +1 hour: 1.3 ± 0.9, +2 hours: 4.1 ± 0.9), peaking at +3 hours (10 ± 1.9 TF mRNA molecules per monocyte). As TF mRNA levels increased, thrombin generation was augmented. Peak levels of TAT and F1 + 2 were reached later (at t +4 hours) than those of TF mRNA. TF mRNA, TAT, and F1 + 2 levels returned to baseline after 24 hours. In conclusion, we used a NASBA/ECL-based technique to quantify TF mRNA in whole blood during human endotoxemia and observed a 125-fold increase in TF mRNA levels. Our data demonstrate a pivotal role for enhanced TF gene activity in the activation of coagulation after LPS challenge.

scholarly journals Production of monoclonal antibodies against Streptococcus mutans antigens

2006 ◽  
Vol 20 (4) ◽  
pp. 297-302 ◽  
Author(s):  
Antonio Carlos Victor Canettieri ◽  
Fujiko Yamasiro Kretchetoff ◽  
Cristiane Yumi Koga-Ito ◽  
Daniella Moreira ◽  
Fabio José Condino Fujarra ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Monoclonal Antibodies ◽  
Streptococcus Mutans ◽  
Cardiac Tissue ◽  
Cross Reactivity ◽  
Oral Streptococci ◽  
Significant Difference ◽  
Common Antigens ◽  
Human Cardiac Tissue

Several studies have been conducted in the last decades aiming to obtain an anti-caries vaccine, however some studies have demonstrated cross reactivity between Streptococcus mutans surface antigens and the human cardiac tissue. In this work, the reactivity of five anti-Streptococcus mutans monoclonal antibodies (MoAb) (24A, 56G, C8, E8 and F6) was tested against oral streptococci, cardiac antigens and skeletal and cardiac myosins, aiming to evaluate the specificity of these MoAb. The hybrid producers of immunoglobulins of the IgG2b class were cloned by limit dilution and expanded in vivo. MoAb were tested by ELISA. The hybrid 24A reacted with S. mutans CCT 1910, S. salivarius CCT 0365 and S. pyogenes T23. No reactivity difference was observed among the tested species. Cross reactivity with heart and cardiac myosin was not confirmed and only reaction with myosin of skeletal muscle was observed (p = 0.0381). The hybrid 56G reacted with all the tested microorganisms and there was statistically significant difference between S. mutans and S. pyogenes T23 (p < 0.001). This hybrid also reacted with myosin of skeletal muscle (p = 0.0095). C8, E8 and F6 presented low reactivity against oral streptococci strains and no reactivity against cardiac antigens. The data of this study showed that the 24A and 56G anti-S. mutans MoAb presented reactivity with S. pyogenes and S. salivarius, reinforcing the occurrence of common antigens between these species. The tested MoAb presented low cross-reactivity with myosin of skeletal muscle, but anti-heart activity could not be confirmed.

Sign in / Sign up

Export Citation Format

Share Document