scholarly journals Alpha 2-macroglobulin-kallikrein complexes detect contact system activation in hereditary angioedema and human sepsis

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2660-2667 ◽  
Author(s):  
N Kaufman ◽  
JD Page ◽  
RA Pixley ◽  
R Schein ◽  
AH Schmaier ◽  
...  
Keyword(s):  
Hereditary Angioedema ◽  
Close Correlation ◽  
Sepsis Syndrome ◽  
Blood Cultures ◽  
Contact System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Sepsis ◽  
Normal Plasma ◽  
System Activation ◽  
High Level

Activation of the contact system has been documented in severe sepsis and hereditary angioedema, but a sensitive, specific, and quantitative assay for assessing the degree of involvement of this proteolytic enzyme cascade is not yet available. We have developed a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) for the alpha 2- macroglobulin-kallikrein (alpha 2M-Kal) complex using an F(ab')2 derivative of a monospecific polyclonal antibody against alpha 2 M as the capture antibody and a unique murine monoclonal antibody, 13G11, against the heavy chain of kallikrein as the detector antibody. The assay does not detect complexes in normal plasma but reacts with complexes generated by activating normal plasma with dextran sulfate at 4 degrees C in a range of 5 to 375 nmol/L. A close correlation of the ELISA with an amidolytic assay for alpha 2M-Kal was documented. Patients with sepsis syndrome but negative bacterial blood cultures did not show elevated plasma complexes, whereas a majority of those with positive blood cultures did show modest elevation and a single patient with septic shock showed a very high level of alpha 2M-Kal complex. Similarly, a patient with classic hereditary angioedema (HAE) showed increased concentration of complexes on three separate occasions during attacks but normal levels between attacks. Two other HAE patients did not show elevated levels at quiescent periods. The ELISA for alpha 2M- Kal appears to be sensitive, specific, and quantitative, and it can be used to reflect the degree of contact system activation in human sepsis and in HAE attacks.

scholarly journals Alpha 2-macroglobulin-kallikrein complexes detect contact system activation in hereditary angioedema and human sepsis

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2660-2667 ◽  
Author(s):  
N Kaufman ◽  
JD Page ◽  
RA Pixley ◽  
R Schein ◽  
AH Schmaier ◽  
...  
Keyword(s):  
Hereditary Angioedema ◽  
Close Correlation ◽  
Sepsis Syndrome ◽  
Blood Cultures ◽  
Contact System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Sepsis ◽  
Normal Plasma ◽  
System Activation ◽  
High Level

Abstract Activation of the contact system has been documented in severe sepsis and hereditary angioedema, but a sensitive, specific, and quantitative assay for assessing the degree of involvement of this proteolytic enzyme cascade is not yet available. We have developed a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) for the alpha 2- macroglobulin-kallikrein (alpha 2M-Kal) complex using an F(ab')2 derivative of a monospecific polyclonal antibody against alpha 2 M as the capture antibody and a unique murine monoclonal antibody, 13G11, against the heavy chain of kallikrein as the detector antibody. The assay does not detect complexes in normal plasma but reacts with complexes generated by activating normal plasma with dextran sulfate at 4 degrees C in a range of 5 to 375 nmol/L. A close correlation of the ELISA with an amidolytic assay for alpha 2M-Kal was documented. Patients with sepsis syndrome but negative bacterial blood cultures did not show elevated plasma complexes, whereas a majority of those with positive blood cultures did show modest elevation and a single patient with septic shock showed a very high level of alpha 2M-Kal complex. Similarly, a patient with classic hereditary angioedema (HAE) showed increased concentration of complexes on three separate occasions during attacks but normal levels between attacks. Two other HAE patients did not show elevated levels at quiescent periods. The ELISA for alpha 2M- Kal appears to be sensitive, specific, and quantitative, and it can be used to reflect the degree of contact system activation in human sepsis and in HAE attacks.

scholarly journals A novel murine in vivo model for acute hereditary angioedema attacks

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sujata Bupp ◽  
Matthew Whittaker ◽  
Mari Lehtimaki ◽  
JuMe Park ◽  
Jessica Dement-Brown ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Hereditary Angioedema ◽  
Silica Nanoparticle ◽  
Ace Inhibition ◽  
Contact System ◽  
In Vivo Model ◽  
System Activation ◽  
The Many ◽  
Deficient Mice

AbstractHereditary Angioedema (HAE) is a rare genetic disease generally caused by deficiency or mutations in the C1-inhibitor gene, SERPING1, a member of the Serpin family. HAE results in acute attacks of edema, vasodilation, GI pain and hypotension. C1INH is a key inhibitor of enzymes controlling complement activation, fibrinolysis and the contact system. In HAE patients, contact system activation leads to uncontrolled production of bradykinin, the vasodilator responsible for the characteristic symptoms of HAE. In this study, we present the first physiological in vivo model to mimic acute HAE attacks. We evaluate hypotension, one of the many hallmark symptoms of acute HAE attacks using Serping1 deficient mice (serping1−/−) and implanted telemetry. Attacks were induced by IV injection of a silica nanoparticle (SiNP) suspension. Blood pressure was measured in real time, in conscious and untethered mice using implanted telemetry. SiNP injection induced a rapid, reversible decrease in blood pressure, in the presence of angiotensin converting enzyme (ACE) inhibition. We also demonstrate that an HAE therapeutic, ecallantide, can prevent HAE attacks in this model. The in vivo murine model described here can facilitate the understanding of acute HAE attacks, support drug development and ultimately contribute to improved patient care.

Contact system activation during erythema marginatum in hereditary angioedema

2020 ◽  
Vol 124 (4) ◽  
pp. 394-395.e1
Author(s):  
Amie Nguyen ◽  
Bruce L. Zuraw ◽  
Sandra C. Christiansen
Keyword(s):  
Hereditary Angioedema ◽  
Contact System ◽  
System Activation

Western Blot Analyses of Prekallikrein and its Activation Products in Human Plasma

1991 ◽  
Vol 65 (04) ◽  
pp. 382-388 ◽  
Author(s):  
Dulce Veloso ◽  
Robert W Colman
Keyword(s):  
Complex Formation ◽  
Western Blot ◽  
Human Plasma ◽  
Contact System ◽  
Plasma Activation ◽  
Normal Plasma ◽  
Sds Page ◽  
Activation Products ◽  
Major Antigen ◽  
Formation Of Complexes

SummaryPrekallikrein (PK), a zymogen of the contact system, and its activation products, kallikrein (KAL), KAl-inhibitor complexes and fragments containing KAL epitope(s) have been detected in human plasma by immunoblotting with a monoclonal anti-human plasma PK antibody, MAb 13G1L. Detection of antigen-MAb 13G11 complexes with peroxidase-conjugated anti-IgG showed that the two variants of PK (85- and 88-kDa) are the only major antigen species in normal, non-activated plasma. Upon plasma activation with kaolin, the intensity of the PK bands decreased with formation of complexes of KAL with CL inhibitor (C1 INH) and α2-rrtzcroglobulin (α2M) identical to those formed by the purified proteins. Immunoblots of normal plasma showed good correlation between the PK detected and the amount of plasma assayed. Increasing amounts of KAL incubated with a constant volume of PK-deficient plasma showed increasing amounts of KAL and of KAL-C1 INH and KAL-α2M complexes. Complexes of KALantithrombin III (ATIII) and the ratio of KALα2M/ KAL-CL INH were higher in activated CL INH-deficient plasmas than in activated normal plasmas. Protein resolution by 3-12% gradient SDS-PAGE and epitope detection with [125I]MAb 13G11 showed four KALα2M species and a 45-kDa fragment(s) in both surface-activated normal plasma and complexes formed by purified KAL and α2M. Immunoblots of activated plasma also showed bands at the position of KALCL INH and KALATIII complexes. When α1-antitrypsin Pittsburgh (cα1-AT, Pitts) was added to plasma before activation, KAL-α1-ALPitts was the main complex. The non-activated normal plasma revealed only an overloaded PK band. This is the first report of an antibody that recognizes KAL epitope(s) in KAL-α2M, KALATIII and KALa1-α1Pitts complexes and in the 45-kDa fragment(s). Therefore, MAb 13G11 should be useful for studying the structure of these complexes as well as the mechanism of complex formation. In addition, immunoblotting with MAb 13G11 would allow detection of KAl-inhibitor complexes in patient plasmas as indicators of activation of the contact system.

scholarly journals AB0103 THE AMBIGUOUS ROLE OF TISSUE CYTOKINES IN THE PATHOGENESIS OF RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1350.1-1351
Author(s):  
O. Korolik ◽  
В. Zavodovsky ◽  
E. Papichev ◽  
Y. Polyakova ◽  
S. L ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
High Activity ◽  
Inflammatory Cytokines ◽  
Stage Iii ◽  
Enzyme Linked Immunosorbent Assay ◽  
Healthy Individuals ◽  
Fetuin A ◽  
High Level ◽  
Extraarticular Manifestations ◽  
Pro Inflammatory Cytokines

Background:Cytokines stimulate the inflammatory response in the synovial membrane with rheumatoid arthritis (RA), initiate apoptosis of chondrocytes, activation of osteoclasts. The progression of comorbid diseases is also associated with the influence of cytokines. At the same time, anti-inflammatory cytokines are produced in various tissues. Their role in the pathogenesis of RA and its complications is ambiguous.Adiponectin (A) and Fetuin A (FA) are classified as negative acute phase proteins. Their concentration decreases with an increase in the level of pro-inflammatory cytokines: TNF-α, IL-1 and IL-6. Molecules A and FA, regardless of various factors and from each other, have similar effects in relation to pro-inflammatory cytokines, lipid and carbohydrate metabolism.Visfatin (V) and Nesfatin-1 (N-1) are pro-inflammatory adipokines. B is produced by cells of the mononuclear phagocytic system and connective tissue. N-1 - is produced by the cells of the intermediate and medulla oblongata and by the cells of the gastric mucosa.Objectives:to study the correlation of B, H-1, A and FA with the severity of inflammation in RAMethods:60 patients with RA and 30 healthy individuals were examined. The level of cytokines was determined by an indirect enzyme-linked immunosorbent assay using commercial test systems (Bio Vendor, cat No. RD195023100, Bio Vendor Human Fetuin-A, RaiBiotech, cat No. EIA-VIS-1, RaiBiotech, cat No. EIA-NESF). All patients underwent a full examination. Diagnosed with 2010 EULAR / ACR recommendations.Results:A decreased level of A (less than 0.8 μg/ml) was detected in 15 patients (25%), F-A (less than 653.55 μg/ml) in 16 (27%), a high level of V (more than 39 ng/ml) - in 55 (91%), N-1 (more than 37.95 ng/ml) - in 36 (60%), which is significantly more often than in healthy individuals. No significant difference in the levels of determined adipokines was found depending on the gender and body weight of patients with RA. The level of cytokines in RA is associated with high activity according to DAS 28, positivity by Anti-CCP, extraarticular manifestations of RA. The greatest correlation with extraarticular manifestations is with cutaneous and cerebral vasculitis. The levels of FA and N-1 also correlated with more pronounced radiological changes (X-ray stage III). FA circulating inhibitor of ectopic calcification. N-1 level is positively correlated with systolic blood pressure.Conclusion:A low level of A and FA, a high level of V and N-1 is characteristic of RA with the presence of high activity and positivity in the RF and Anti-CCP. An increased level of B is determined by more than 90% of patients, which indicates its high pro-inflammatory activity. The level of F and N-1 is also associated with the degree of damage to bone tissue (stage III, a lot of erosion). A positive correlation of level V and N-1, negative A and FA with the severity of inflammation in RA confirms the involvement of these proteins in the pathogenesis. A high level of A and V increases the risk of developing cardiovascular diseases and their complications, the effect of N-1 and FA is being studied. The effect of cytokines on osteoclasts and osteoblasts in RA is ambiguousReferences:[1]Visfatin and Rheumatoid Arthritis: Pathogenetic Implications and Clinical Utility. Polyakova Y. Curr Rheumatol Rev.2019[2]Serum nesfatin -1 as a marker of systemic inflammation in rheumatoid arthritis. Kvlividze T. Klinicheskaya Laboratornaya Diagnostika.2019; 64 (1):53-56 (in Russ)[3]Fetuin-A. Novel hepatokine in rheumatoid arthritis laboratory diagnostics. Papichev E. Klinicheskaya Laboratornaya Diagnostika.2018; 63 (12):756-760 (in Russ)Disclosure of Interests:None declared

scholarly journals Contact system activation and high thrombin generation in hyperthyroidism

2017 ◽  
Vol 176 (5) ◽  
pp. 583-589 ◽  
Author(s):  
Namhee Kim ◽  
Ja-Yoon Gu ◽  
Hyun Ju Yoo ◽  
Se Eun Han ◽  
Young Il Kim ◽  
...  
Keyword(s):  
Neutrophil Elastase ◽  
Thrombin Generation ◽  
Contact System ◽  
Factor Vii ◽  
Factor Xii ◽  
Free T4 ◽  
Thrombotic Risk ◽  
Double Stranded Dna ◽  
System Activation ◽  
Normal Controls

BackgroundHyperthyroidism is associated with increased thrombotic risk. As contact system activation through formation of neutrophil extracellular traps (NET) has emerged as an important trigger of thrombosis, we hypothesized that the contact system is activated along with active NET formation in hyperthyroidism and that their markers correlate with disease severity.Subjects and methodsIn 61 patients with hyperthyroidism and 40 normal controls, the levels of coagulation factors (fibrinogen, and factor VII, VIII, IX, XI and XII),D-dimer, thrombin generation assay (TGA) markers, NET formation markers (histone–DNA complex, double-stranded DNA and neutrophil elastase) and contact system markers (activated factor XII (XIIa), high-molecular-weight kininogen (HMWK), prekallikrein and bradykinin) were measured.ResultsPatients with hyperthyroidism showed higher levels of fibrinogen (median (interquartile range), 315 (280–344) vs 262 (223–300),P = 0.001),D-dimer (103.8 (64.8–151.5) vs 50.7 (37.4–76.0),P < 0.001), peak thrombin (131.9 (102.2–159.4) vs 31.6 (14.8–83.7),P < 0.001) and endogenous thrombin potential (649 (538–736) vs 367 (197–1147),P = 0.021) in TGA with 1 pM tissue factor, neutrophil elastase (1.10 (0.39–2.18) vs 0.23 (0.20–0.35),P < 0.001), factor XIIa (66.9 (52.8–87.0) vs 73.0 (57.1–86.6),P < 0.001), HMWK (6.11 (4.95–7.98) vs 3.83 (2.60–5.68),P < 0.001), prekallikrein (2.15 (1.00–6.36) vs 1.41 (0.63–2.22),P = 0.026) and bradykinin (152.4 (137.6–180.4) vs 118.3 (97.1–137.9),P < 0.001) than did normal controls. In age- and sex-adjusted logistic regression analysis, fibrinogen, factor VIII, IX and XIIa,D-dimer, peak thrombin, neutrophil elastase, HMWK and bradykinin showed significant odds ratios representing hyperthyroidism’s contribution to coagulation and contact system activation. Free T4 was significantly correlated with factors VIII and IX,D-dimer, double-stranded DNA and bradykinin.ConclusionThis study demonstrated that contact system activation and abundant NET formation occurred in the high thrombin generation state in hyperthyroidism and were correlated with free T4 level.

scholarly journals Polyphosphate nanoparticles on the platelet surface trigger contact system activation

Blood ◽  
2017 ◽  
Vol 129 (12) ◽  
pp. 1707-1717 ◽  
Author(s):  
Johan J. F. Verhoef ◽  
Arjan D. Barendrecht ◽  
Katrin F. Nickel ◽  
Kim Dijkxhoorn ◽  
Ellinor Kenne ◽  
...  
Keyword(s):  
Metal Ions ◽  
Divalent Metal ◽  
Contact System ◽  
Divalent Metal Ions ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Key Points ◽  
System Activation

Key Points Activated platelets expose insoluble membrane-associated polyphosphate nanoparticles that are complexed with divalent metal ions. Platelet polyphosphate nanoparticles, but not soluble polyphosphate polymers, activate the contact system.

scholarly journals The frequency of resistance to antibiotics of most frequently isolated bacteria from blood cultures during the period 1997-2002

2004 ◽  
Vol 61 (4) ◽  
pp. 391-397
Author(s):  
Veljko Mirovic ◽  
Branka Tomanovic ◽  
Sonja Konstantinovic
Keyword(s):  
Clinical Laboratory ◽  
Blood Cultures ◽  
Diffusion Method ◽  
National Committee ◽  
Disk Diffusion Method ◽  
Beta Lactamases ◽  
Resistance To Antibiotics ◽  
Extended Spectrum Beta Lactamases ◽  
High Level

The aim of this study was to determine the frequency of resistance to antibiotics of the most frequently isolated bacteria from blood cultures of hospitalized patients during the period 1997-2002. The resistance to antibiotics was determined by disk diffusion method according to National Committee for Clinical Laboratory Standards procedures. The majority of staphylococci isolates were resistant to methicillin, and the proportion of methicillin-resistant Staphylococcus aureus was stable (76.8-81.6%), during the follow-up period. None of the staphylococci isolates were resistant to vancomycin, but there was a very high incidence of high-level resistance of enterococci to aminoglycosides (47.2-72.2%). In 1998, only one strain among enterococci was resistant to vancomycin (Enterococcus faecium, VanA fenotype). Enterococcus spp isolates expressed variable frequency of resistance to ampicillin (15-40.1%) during the follow-up period. Among Enterobacteriaceae there were no isolates resistant to imipenem, but dramatic increase of the resistance to ceftriaxone was found from 35.9% in 1997 to 95.9% in 2002 (p<0.001). Extended spectrum beta-lactamases production was found in all the species of enterobacteria isolates. Resistance to imipenem was observed in Acinetobacter spp isolates in 2002 for the first time. Pseudomonas spp isolates expressed high and very variable resistance to all antibiotics tested during the follow-up period.

scholarly journals High-Level Expression of Plasmodium vivax Apical Membrane Antigen 1 (AMA-1) in Pichia pastoris: Strong Immunogenicity in Macaca mulatta Immunized with P. vivax AMA-1 and Adjuvant SBAS2

1999 ◽  
Vol 67 (1) ◽  
pp. 43-49 ◽  
Author(s):  
Clemens H. M. Kocken ◽  
Martin A. Dubbeld ◽  
Annemarie Van Der Wel ◽  
Jack T. Pronk ◽  
Andrew P. Waters ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Plasmodium Vivax ◽  
Apical Membrane ◽  
Gel Filtration ◽  
Methylotrophic Yeast ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
High Level

ABSTRACT The apical membrane antigen 1 (AMA-1) family is a promising family of malaria blood-stage vaccine candidates that have induced protection in rodent and nonhuman primate models of malaria. Correct conformation of the protein appears to be essential for the induction of parasite-inhibitory responses, and these responses appear to be primarily antibody mediated. Here we describe for the first time high-level secreted expression (over 50 mg/liter) of thePlasmodium vivax AMA-1 (PV66/AMA-1) ectodomain by using the methylotrophic yeast Pichia pastoris. To prevent nonnative glycosylation, a conservatively mutagenized PV66/AMA-1 gene (PV66Δglyc) lacking N-glycosylation sites was also developed. Expression of the PV66Δglyc ectodomain yielded similar levels of a homogeneous product that was nonglycosylated and was readily purified by ion-exchange and gel filtration chromatographies. Recombinant PV66Δglyc43–487 was reactive with conformation-dependent monoclonal antibodies. With the SBAS2 adjuvant,Pichia-expressed PV66Δglyc43–487 was highly immunogenic in five rhesus monkeys, inducing immunoglobulin G enzyme-linked immunosorbent assay titers in excess of 1:200,000. This group of monkeys had a weak trend showing lower cumulative parasite loads following a Plasmodium cynomolgi infection than in the control group.

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