scholarly journals Activation of Coagulation by Administration of Recombinant Factor VIIa Elicits Interleukin 6 (IL-6) and IL-8 Release in Healthy Human Subjects

2003 ◽  
Vol 10 (3) ◽  
pp. 495-497 ◽  
Author(s):  
Evert de Jonge ◽  
Philip W. Friederich ◽  
George P. Vlasuk ◽  
William E. Rote ◽  
Margaretha B. Vroom ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Interleukin 6 ◽  
Recombinant Factor Viia ◽  
Factor Viia ◽  
Human Subjects ◽  
In Vitro Experiments ◽  
Healthy Human ◽  
Healthy Human Subjects ◽  
Proinflammatory Responses

ABSTRACT The activation of coagulation has been shown to contribute to proinflammatory responses in animal and in vitro experiments. Here we report that the activation of coagulation in healthy human subjects by the administration of recombinant factor VIIa also elicits a small but significant increase in the concentrations of interleukin 6 (IL-6) and IL-8 in plasma. This increase was absent when the subjects were pretreated with recombinant nematode anticoagulant protein c2, the inhibitor of tissue factor-factor VIIa.

scholarly journals Recombinant Human IL-12 Exerts Differential Effects on Circulating CD56bright and CD56dim NK Cells in Healthy Human Subjects

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 756-756
Author(s):  
Simmy Thomas ◽  
Chris E Lawrence ◽  
Vernon Mar ◽  
Hue Kha ◽  
Lena A Basile
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Human Subjects ◽  
Cell Populations ◽  
Employment Equity ◽  
Healthy Human ◽  
Healthy Human Subjects ◽  
Equity Ownership

Abstract Interleukin-12 (IL-12) has potent immunoregulatory and hematopoietic properties, and exerts significant biological effects on natural killer (NK) cells, inducing IFNγ production and enhancing cytotoxicity. Two distinct NK cell populations correlate with their immunoregulatory functions. Mature CD56dimCD16bright NK cells represent 90% of the NK cells resident in the blood and can exert cytotoxic effects on transformed cells. Cytokine producing immature CD56brightCD16+/- NK cells exist in the blood (10% of total circulating NK cells) but are most prominently located in secondary lymphoid tissues. In the continued clinical development of recombinant human IL-12 (HemaMax™, rHuIL-12), to be used in combination with radiotherapy or chemotherapy for the treatment of cancer patients, we have performed a clinical safety study in healthy human subjects. A single subcutaneous (sc) dose of rHuIL-12 (12μg) was administered to 17 healthy human subjects. Placebo was administered to 5 healthy subjects. Peripheral blood samples were collected before rHuIL-12 administration, and up to Day 14 post administration. Immunophenotyping of blood cell populations was conducted by FACS. rHuIL-12 caused a transient decrease in peripheral blood CD56dimCD16bright NK cells, with a nadir (60% reduction from baseline) reached on Day 2 following rHuIL-12 administration. CD56dimCD16bright NK cell levels returned almost to baseline levels on Day 5. Placebo was without effect. Conversely rHuIL-12 caused an elevation in peripheral blood CD56brightCD16+/- NK cells, particularly between Days 2 and 3 after rHuIL-12 administration, which was sustained until a peak was reached on Day 5 (265% above baseline). Levels returned to baseline by Day 11, while placebo was without effect. rHuIL-12 did not impact the less functional CD56-CD16bright NK cell subset. CD56dimCD16bright NK cells expressing the IL-12 receptor β2 subunit (IL-12Rβ2+) showed a substantial, and transient, decrease in levels on Day 2. The plasma concentration of IFNγ was elevated to a peak over 35 fold above baseline level at 10hr. after rHuIL-12 administration. Human NK cells were negatively selected from highly enriched leukapheresis-derived blood and stimulated in vitro with 10 pM rHuIL-12. After 16hr. incubation these predominantly CD56dimCD16brightNK cells showed enhanced release of IFNγ and the increased killing of K562 cells, a human erythroleukemic cell line, when compared with vehicle controls. qPCR analysis of the human NK cell lysates showed rHuIL-12-induced elevation of CD56 (302%) and IL-12Rβ2 (587%) mRNA, when compared with vehicle controls. rHuIL-12 did not influence CD16 mRNA expression, but did increase the level of CD62L (L selectin, 206%) mRNA. The rapid 60% fall in circulating mature CD56dimCD16bright NK cells after rHuIL-12 administration to healthy human subjects suggests their immediate exit from peripheral blood into the tissue compartments. This could be mediated by the observed increase in NK cell CD62L mRNA expression seen in vitro. The sustained increase in immature CD56brightCD16+/- NK cell levels between Day 3 and 6 suggests their IL-12-induced development from CD34+ hematopoietic progenitor cells. In summary rHuIL-12 administration to healthy human subjects demonstrates differential effects on the two key NK cell populations in peripheral blood, increasing CD56brightCD16+/- NK cell numbers, potentially stimulating IFNγ release from and enhancing the cytotoxicity of the CD56dimCD16bright NK cells, and preparing this population for migration into tissues. rHuIL-12 thus shows excellent potential as an immunotherapeutic and hematopoietic agent for the treatment of cancer patients, by impacting the maturation, activation, immunoregulation, and cytolytic properties of NK cells. Disclosures Thomas: Neumedicines: Employment, Equity Ownership. Lawrence:Neumedicines: Employment, Equity Ownership. Mar:Neumedicines: Employment, Equity Ownership. Kha:Neumedicines: Employment, Equity Ownership. Basile:Neumedicines: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals A randomised, double- blind, cross-over study investigating the prebiotic effect of agave fructans in healthy human subjects

2015 ◽  
Vol 4 ◽  
Author(s):  
P. Ramnani ◽  
A. Costabile ◽  
A. G. R. Bustillo ◽  
G. R. Gibson
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Human Subjects ◽  
Secretory Iga ◽  
Healthy Human ◽  
Double Blind ◽  
Bacterial Populations ◽  
Healthy Human Subjects ◽  
Gradient Gel Electrophoresis

AbstractThis placebo-controlled, randomised, double-blind, cross-over human feeding study aimed to determine the prebiotic effect of agave fructans. A total of thirty-eight volunteers completed this trial. The treatment consisted of 3 weeks' supplementation with 5 g/d of prebiotic agave fructan (Predilife) or equivalent placebo (maltodextrin), followed by a 2-week washout period following which subjects were crossed over to alternate the treatment arm for 3 weeks followed by a 2-week washout. Faecal samples were collected at baseline, on the last day of treatment (days 22 and 58) and washout (days 36 and 72), respectively. Changes in faecal bacterial populations, SCFA and secretory IgA were assessed using fluorescentin situhybridisation, GC and ELISA, respectively. Bowel movements, stool consistencies, abdominal comfort and mood changes were evaluated by a recorded daily questionnaire. In parallel, the effect of agave fructans on different regions of the colon using a three-stage continuous culture simulator was studied. Predilife significantly increased faecal bifidobacteria (log109·6 (sd0·4)) and lactobacilli (log107·7 (sd0·8)) compared with placebo (log109·2 (sd0·4);P = 0·00) (log107·4 (sd0·7);P= 0·000), respectively. No change was observed for other bacterial groups tested, SCFA, secretory IgA, and PGE2concentrations between the treatment and placebo. Denaturing gradient gel electrophoresis analysis indicated that bacterial communities were randomly dispersed and no significant differences were observed between Predilife and placebo treatments. Thein vitromodels showed similar increases in bifidobacterial and lactobacilli populations to that observed with thein vivotrial. To conclude, agave fructans are well tolerated in healthy human subjects and increased bifidobacteria and lactobacilli numbersin vitroandin vivobut did not influence other products of fermentation.

Abstract 420: Exploring the Role of TNFα in TLR4-/NLPR3 Inflammasome-driven Inflammation in Humans

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Matthijs Moerland ◽  
Karen Malone ◽  
Marlous Dillingh ◽  
Wieke Grievink ◽  
Joannes Reijers ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Human Subjects ◽  
Healthy Human ◽  
Mechanistic Investigation ◽  
Healthy Human Subjects ◽  
Key Factor ◽  
Culture Supernatants

The role of TNFα in the pathogenesis of atherosclerosis is incompletely understood. TNFα blockade reduces the severity of various autoimmune diseases and the often related atherosclerosis. However, excessively released TNFα is only one component of the hyperactive innate immune system in such diseases. To provide more insight into the role of TNFα in the induction of inflammation, we explored the effects of TNFα blockade in human whole blood. TLR4/NLPR3 inflammasome challenges were applied to induce an inflammatory response. For this purpose, whole blood was incubated 4 hours with LPS and aluminium hydroxide (Alhydrogel). TNFα blockade was evaluated in vitro (LPS/Alhydrogel challenge in whole blood of 4 healthy human subjects, +concentration range of adalimumab) and ex vivo (LPS/Alhydrogel challenge in whole blood of 13 healthy human subjects receiving a single subcutaneous (sc) dose of 40 mg adalimumab). Cytokine release was evaluated in culture supernatants. In vitro, TNFα blockade strongly reduced TNFα levels detected; -97±1% at the lowest adalimumab concentration (0.3125 μg/mL). TNFα blockade did not affect LPS/Alhydrogel-induced IL-6, IL-1β and IL-18 release, but reduced IFNγ release; maximally -93±4% at 5 μg/mL adalimumab. A single sc adalimumab dose in healthy subjects reduced LPS/Alhydrogel-induced TNFα levels (maximally -98±1% on day 4, and still -58±59% on day 64; versus baseline). IL-6, IL-1β and IL-8 release were not reduced after anti-TNFα treatment. The effect of TNFα blockade on IFNγ release could not be reliably estimated due to highly variable IFNγ levels, especially between genders (baseline IFNγ levels 1248±1771 and 140±283 pg/mL, males vs females). TNFα is a major inducer of NFκB-driven cytokine gene transcription, but TNFα blocking did not reduce LPS/Alhydrogel-induced release of IL-1β, IL-6, IL-8 or IL-18 by primary human cells. This suggests that primary TLR4- and inflammasome-mediated signalling is sufficient to drive secretion of these cytokines. However, in vitro TNFα blockade did impair IFNγ release. Since IFNγ is a key factor in atherogenesis, exerting both pro- and anti-atherogenic properties, our data warrant further mechanistic investigation of the role of TNFα and anti-TNFα therapies in atherosclerosis.

Influence of Age on Polymorphonuclear Leukocytes in vitro: Phagocytic Activity in Healthy Human Subjects

Gerontology ◽  
1986 ◽  
Vol 32 (6) ◽  
pp. 308-316 ◽  
Author(s):  
Giorgio Emanuelli ◽  
Mario Lanzio ◽  
Teresa Anfossi ◽  
Silvia Romano ◽  
Giovanni Anfossi ◽  
...  
Keyword(s):  
Phagocytic Activity ◽  
Polymorphonuclear Leukocytes ◽  
Human Subjects ◽  
Healthy Human ◽  
Healthy Human Subjects

scholarly journals IgG1 Thioether Bond Formation in Vivo

2013 ◽  
Vol 288 (23) ◽  
pp. 16371-16382 ◽  
Author(s):  
Qingchun Zhang ◽  
Matthew R. Schenauer ◽  
John D. McCarter ◽  
Gregory C. Flynn
Keyword(s):  
Light Chain ◽  
Reaction Rate ◽  
Human Subjects ◽  
Light Chains ◽  
Chain Reaction ◽  
Healthy Human ◽  
Conversion Rates ◽  
Healthy Human Subjects

During either production or storage, the LC214-HC220 disulfide in therapeutic antibodies can convert to a thioether bond. Here we report that a thioether forms at the same position on antibodies in vivo. An IgG1κ therapeutic antibody dosed in humans formed a thioether at this position at a rate of about 0.1%/day while circulating in blood. Thioether modifications were also found at this position in endogenous antibodies isolated from healthy human subjects, at levels consistent with this conversion rate. For both endogenous antibodies and recombinant antibodies studied in vivo, thioether conversion rates were faster for IgG1 antibodies containing λ light chains than those containing κ light chains. These light chain reaction rate differences were replicated in vitro. Additional mechanistic studies showed that base-catalyzed thioether formation through the light chain dehydrogenation was more preferred on antibodies with λ light chains, which may help explain the observed reaction rate differences.

Microparticle-associated vascular adhesion molecule-1 and tissue factor follow a circadian rhythm in healthy human subjects

2008 ◽  
Vol 99 (11) ◽  
pp. 909-915 ◽  
Author(s):  
Rebecca Vince ◽  
Marie Sandström ◽  
Lee Taylor ◽  
Lars McNaughton ◽  
Gerard Laden ◽  
...  
Keyword(s):  
Circadian Rhythm ◽  
Tissue Factor ◽  
Adhesion Molecule ◽  
Human Subjects ◽  
Coagulation Cascade ◽  
Healthy Human ◽  
Risk Of Death ◽  
Increased Risk ◽  
Healthy Human Subjects

SummaryAn increased risk of death or severe injury due to late-morning thrombotic events is well established. Tissue factor (TF) is the initiator of the coagulation cascade, and endothelial stresses, coupled with production of pro-coagulant microparticles (MP) are also important factors in loss of haemostasis. TF and vascular cell adhesion molecule-1 (VCAM-1) -positive cell microparticles were assessed periodically over a 24-hour (h) period in healthy human subjects to ascertain if they followed a circadian rhythm. Eleven healthy male subjects were assessed in a temperature-controlled environment with dietary intake consistent between subjects. Blood samples were taken every 4 h by venipuncture, and TF and VCAM-1 positive microparticles were quantified by flow cytometry. A significant circadian rhythm was observed in VCAM-1 MP (p=<0.0001), and a trend was shown, although not statistically significant (p=0.065) in TF microparticles. A peak was observed at 9 a.m. for VCAM-1 positive MP, followed by a decrease and subsequent peak at 9 p.m. and a minimum at 5 a.m. TF-positive MP followed a strikingly similar trend in both variation and absolute numbers with a delay. A circadian rhythm was observed in VCAM-1 and less so TF-positive MP. This has significant implications in terms of the well known increased risk of cardiovascular thrombotic events matching this data. To our knowledge this is the first such report of quantified measurements of these MP over a 24-h period and the only measurement of a 24-h variation of in-vivo blood-borne TF.

Reversal Of Dabigatran By Recombinant Factor VIIa Is Dependent On The Amount Of Tissue-Factor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1149-1149 ◽  
Author(s):  
Xena X Li ◽  
Ivan Stevic ◽  
Frank M.H. Lee ◽  
Keith K. Lau ◽  
Anthony K.C. Chan ◽  
...  
Keyword(s):  
Animal Models ◽  
Tissue Factor ◽  
Recombinant Factor Viia ◽  
Conflicts Of Interest ◽  
Factor Viia ◽  
In Vivo Studies ◽  
Bleeding Complications ◽  
Hemostatic Agents

Abstract Background Dabigatran is a new oral anticoagulant that specifically and reversibly inhibits thrombin. Its predictable pharmacokinetics and pharmacodynamics allow for minimal monitoring. However, there is currently no specific antidote to reverse its anticoagulant effects. Instead, activated prothrombin complex concentrate (aPCC) or recombinant Factor VIIa (rFVIIa) has been used to stop bleeding complications in patients on dabigatran. Both were originally used to treat hemophilia patients with inhibitors. Currently, Factor Eight Inhibitor Bypass Activity (FEIBA) is the only clinically approved aPCC, which contains Factors II, IX, X, and VII in both active and non-active forms. In contrast, rFVIIa is a human recombinant protein which can initiate clotting via the tissue factor (TF) pathway. It has been controversial which of these hemostatic agents is more efficient at reversing the effect of dabigatran, as animal models have failed to yield consistent results. We hereby utilized a modified Hemoclot turbdity assay to determine the equivalent concentrations of FEIBA and rFVIIa that can reverse the anticoagulant effect of dabigatran. Methods A mixture was prepared by incubating normal pooled plasma (NPP) with 382 nM dabigatran and varying concentration of hemostatic agents (FEIBA or rFVIIa), in the absence or presence of TF (Thrombosel®). The mixture was diluted 1:8 in TSP buffer, from which a 50 µL-aliquot was then incubated with 100 µL of NPP at 37°C for 5 min. Clotting was initiated with 100 µL of another mixture containing 10 mM Ca2+ and 2.5 nM thrombin in TSP. Turbidity was measured at 350 nm using a SpectraMax Pro spectrophotometer at 37°C for 2 hr. Clotting time (CT) was defined as the time to reach half of the maximum turbidity. Results In this modified Hemoclot turbidity assay, the CT without dabigatran was 101 s and it linearly increased dependent on the dabigatran concentration up to 1500 nM. Dabigatran at 382 nM, the therapeutic plasma concentration, prolonged CT 5-fold to 505 s. Addition of 1 U/mL FEIBA reduced the CT approximately 35% to 328 s. The reversal effect of FEIBA plateaued at 2.5 U/mL to 5 U/mL because there was minimal further reduction in the CT even with 10 U/mL FEIBA. In contrast, rFVIIa at a therapeutic concentration of 50 nM barely reduced the CT by <10% to 469 s. The reversal effects of rFVIIa were drastically enhanced by the addition of extrinsic tissue factor. With TF at 1.5 pM, the rFVIIa reduced the CT to a level similarly achieved by 1 U/mL of FEIBA, but the CT could not be reduced further despite the concentration of rFVIIa increased 10-fold to 400 nM. Finally, although both FEIBA and rFVIIa/TF reached an eventual plateau in the reversal of dabigatran, none of them could lower the CT to the baseline level. Summary/Conclusions Our in vitro study shows that reversal of dabigatran by rFVIIa is dependent on the concentration of TF. Higher levels of TF augment the reversal effects of rFVIIa to the extents similarly achieved with FEIBA. The data may, in part, explain the inconsistency of results obtained from in vivo studies using various animal models to compare rFVIIa and FEIBA for the reversal of dabigatran. The circulating TF levels in vivo are much lower than the amount of TF used in this study, thus its availability in the microenvironment for hemostasis varies depending on the methods used to induce bleeding. The data in this study also explain the variable clinical efficacy of rFVIIa reported in the literature when it is used to reverse the bleeding complications in patients on dabigatran. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In vitro evidence of a tissue factor-independent mode of action of recombinant factor VIIa in hemophilia

Blood ◽  
2014 ◽  
Vol 124 (20) ◽  
pp. 3172-3174 ◽  
Author(s):  
Cecilia Augustsson ◽  
Egon Persson
Keyword(s):  
Tissue Factor ◽  
Mode Of Action ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Recombinant Factor Viia ◽  
Negative Impact ◽  
Factor Viia ◽  
Key Points ◽  
Independent Mode

Key Points The negative impact on thrombin generation of zymogen FVII competing with rFVIIa for TF is counteracted by FVII (auto)activation. Correction of hemophilia A occurs in a rFVIIa concentration range where detectable effects of FVII competition are minimal or absent.

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