scholarly journals Sample Aging Profoundly Reduces Monocyte Responses in Human Whole Blood Cultures

2018 ◽  
Vol 2018 ◽  
pp. 1-9
Author(s):  
H. W. Grievink ◽  
M. Moerland
Keyword(s):  
Whole Blood ◽  
Intracellular Cytokine Staining ◽  
Drug Effects ◽  
Blood Cultures ◽  
Blood Collection ◽  
Innate Immune Responses ◽  
Cytokine Release ◽  
Human Whole Blood ◽  
The Public

Human whole blood cultures are widely used for the investigation of physiological pathways and drug effectsin vitro. Detailed information on the effect of “sample aging” (the time span between blood collection and experimental start) on the experimental outcome is not readily available in the public domain. We studied the effect of sample aging on the ability of immune cells to respond to cell-specific immune triggers (LPS, PMA/ionomycin, and SEB). Sample aging at room temperature profoundly inhibited the LPS-induced monocytic cytokine release in minimally diluted whole blood cultures. The reduction ranged from 20–50% after 30 minutes to 80–100% after 10 hours and differed between cytokines (IL-1β, IL-2, IL-6, IFNγ, and TNFα). Sample storage at 4°C or 37°C even worsened this. PMA/ionomycin- and SEB-induced cytokine release, both mainly T-cell-driven, were also reduced by sample aging but to a lesser extent (20–50% after 24 hours). Intracellular cytokine staining revealed that the number of LPS-responding cells was not impacted by sample aging and reduced LPS responsivity could also not be explained by apoptosis or downregulated TLR4 expression. Thus, we speculate that sample aging induces an inhibitory pathway downstream from TLR4 in monocytes. These results underline the importance of quick sample handling when investigating innate immune responses in whole blood, especially for monocyte responses.

scholarly journals Evaluation of the In Vitro Pyrogen Test System Based on Proinflammatory Cytokine Release from Human Monocytes: Comparison with a Human Whole Blood Culture Test System and with the Rabbit Pyrogen Test

2002 ◽  
Vol 9 (3) ◽  
pp. 588-597 ◽  
Author(s):  
Yukari Nakagawa ◽  
Hideko Maeda ◽  
Toshimi Murai
Keyword(s):  
Blood Culture ◽  
Whole Blood ◽  
Proinflammatory Cytokine ◽  
Ex Vivo ◽  
Test System ◽  
In Vitro Test ◽  
Cytokine Release ◽  
Human Whole Blood ◽  
Culture Test

ABSTRACT The reliability of an in vitro pyrogen test system based on proinflammatory cytokine release from human monocytic cells was assessed by comparison with a test system based on a human whole blood culture as well as with the conventional rabbit pyrogen test. The human cells used as the pyrogen indicator cells were newly selected by subcloning of a human monocytic cell line, Mono-Mac-6. The selected cells, named MM6-CA8, responded to various pyrogens, including endotoxin, peptidoglycan (PG), Staphylococcus aureus Cowan 1 (SAC), and poly(I ·  C), with a high sensitivity and produced proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha. Among these cytokines, IL-6 was produced most sensitively in response to traces of the pyrogens and detected in the largest quantities in the culture medium. The cytokine-producing responses of MM6-CA8 cells correlated significantly with the responses of cultured human whole blood, which represents an ex vivo culture test system reproducing pyrogen-induced cytokine production in the human body. In terms of cytokine inducibility, the pyrogens were ranked in the order endotoxin > PG > poly (I · C) > SAC in both culture systems, a ranking which almost agreed with the ranking of their pyrogenicity as assessed by the rabbit pyrogen test. These results suggest that the in vitro responsiveness of MM6-CA8 cells to various pyrogens is highly relevant for human pyrogenic reactions. Therefore, the in vitro test system is useful and reliable for detecting the presence of materials that are pyrogenic for humans.

Gravity-Related Immunological Changes in Human Whole Blood Cultured Under Simulated Microgravity Using an In Vitro Cytokine Release Assay

2017 ◽  
Vol 37 (12) ◽  
pp. 531-540 ◽  
Author(s):  
Merel Van Walleghem ◽  
Kevin Tabury ◽  
Rodrigo Fernandez-Gonzalo ◽  
Ann Janssen ◽  
Judith-Irina Buchheim ◽  
...  
Keyword(s):  
Whole Blood ◽  
Simulated Microgravity ◽  
Cytokine Release ◽  
Human Whole Blood ◽  
Release Assay ◽  
Immunological Changes

scholarly journals Importance of Preanalytical Factors in Measuring Cr and Co Levels in Human Whole Blood: Contamination Control, Proper Sample Collection and Long-Term Storage Stability

2020 ◽  
Author(s):  
Yuliya L Sommer ◽  
Cynthia D Ward ◽  
Joaudimir Castro Georgi ◽  
Po-Yung Cheng ◽  
Robert L Jones
Keyword(s):  
Stainless Steel ◽  
Whole Blood ◽  
Consumer Products ◽  
Blood Collection ◽  
Sample Collection ◽  
Blood Samples ◽  
Contamination Control ◽  
Human Whole Blood

Abstract A number of errors with potentially significant consequences may be introduced at various points in the analytical process, which result in skewed, erroneous analytical results. Precautionary procedures such as contamination control, following established sample collection protocols, and having a complete understanding of the long-term stability of the elements of interest can minimize or eliminate these errors. Contamination control is critical in the quantification of Cr and Co in human whole blood. Cr and Co levels in most biological samples are low, but these elements occur naturally in the environment and are often found in commercial and consumer products, which increases the risk of contamination. In this paper, we demonstrated that lot screening process in which we pre-screen a sub-set of manufactured lots used in collecting, analyzing and storing blood samples is a critical step in controlling Cr and Co contamination. Stainless steel needles are often utilized in blood collection but are considered as a potential source of introducing metal contamination to the patient sample. We conducted two studies to determine if there is a possibility of Cr or Co leaching into the human whole blood from the needles during blood collection. We analyzed blood collected from 100 donors and blood collected in vitro in the laboratory from designated vessel containing spiked blood with higher levels of Cr and Co. Two blood tubes were consecutively collected through one needle. In both studies, Cr and Co concentration levels in the two consecutively collected tubes were compared. Based on the results from donor and in vitro blood collection studies, we concluded that there was no Cr and Co leaching from the limited sets of stainless steel needles used in these studies. Furthermore, we demonstrated that Cr and Co human whole blood samples are stable for 1 year stored at temperatures of −70, −20 and 4°C and 6 months at room temperature.

Elotuzumab (HuLuc63) Activates CD56dim Natural Killer Cells and Monocytes Resulting in the Release of IP-10 and MCP-1

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 108-108 ◽  
Author(s):  
Balaji Balasa ◽  
Mahrukh Huseni ◽  
Jyothi Cherukuri ◽  
Roxanne Steinle ◽  
Amulya Nanisetti ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Nk Cells ◽  
Whole Blood ◽  
Cell Activation ◽  
Nk Cell ◽  
Blood Cultures ◽  
Monocyte Activation ◽  
Human Whole Blood ◽  
Low Levels

Abstract Elotuzumab (formerly known as HuLuc63) is a humanized monoclonal antibody targeting CS1 (CD319, SLAMF7), a protein which is highly and uniformly expressed on myeloma cells. CS1 is also expressed at lower levels on selected lymphocyte subsets in humans, including natural killer (NK) cells and a subset of CD8+ T cells. Elotuzumab mediates killing of multiple myeloma cell lines (OPM-2, L363) via antibody-dependent cellular cytotoxicity in vitro and has significant anti-tumor activity in mouse xenograft models. Elotuzumab is currently being investigated in Phase I safety studies in relapsed multiple myeloma (MM). To further characterize the effects of elotuzumab on different immune cell subsets, we evaluated its effects in human whole blood cultures in vitro. In resting human whole blood, >95% of the CD56dim (CD3−CD56+CD16++) NK population expressed CS1. In contrast, only 50–75% of CD56bright NK cells expressed CS1. The expression level of CS1, as measured by mean fluorescence intensity, was also lower in CD56bright than in CD56dim NK cells. In 24-hr whole blood cultures, elotuzumab selectively activated CD56dim NK cells, as evidenced by up-regulation of CD69, CD11b, CD54, and down-modulation of CD16 expression, while having little or no effect on CD56bright NK cells. Monocytes in the same whole blood culture were also activated as determined by up-regulation of CS1, HLA-DR, and CD54. Monocyte activation was significantly correlated with CD56dim NK cell activation. In contrast, elotuzumab induced little to no activation of either CD4+ or CD8+ T cell subsets. In whole blood cultures, elotuzumab induced low levels of IFN-γ and only negligible levels of TNF-α. However, elotuzumab induced robust levels of the chemokines IP-10 and MCP-1 which correlated significantly with the level of CD56dim NK cell activation. The induction of chemokines was both dose and Fc-dependent, as F(ab′)2 fragments of elotuzumab failed to induce either NK cell activation or release of chemokines. Interestingly, when elotuzumab was added to purified NK cell cultures, no increase in the levels of IP-10 and MCP-1 was observed. Addition of neutralizating anti-IFN-g mAb to elotuzumab-treated whole blood cultures had little effect on NK cell activation but partially reduced monocyte activation and IP-10 release. Finally, addition of anti-CD18 mAb abrogated the elotuzumab-mediated activation of NK cells and monocytes suggesting cell-cell contact plays an important role in elotuzumab-induced activation of CD56dim NK cells and monocytes. In conclusion, the result from this study demonstrated that elotuzumab may activate NK cells via CS1 and CD16 signaling, resulting in the release of low levels of IFN-g and subsequently monocyte-dependant chemokine production including IP-10 and MCP-1. When dosed in a phase I clinical trial, elotuzumab induced a transient elevation of the chemokines IP-10 and MCP-1 in the serum of MM patients, indicating that the whole blood culture system enables the identification of appropriate pharmacodynamic markers.

scholarly journals AB0095 PRECLINICAL CHARACTERIZATION OF CJ-15314, A HIGHLY SELECTIVE JAK1 INHIBITOR, FOR THE TREATMENT OF AUTOIMMUNE DISEASES

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1347.2-1347
Author(s):  
S. Y. Ki ◽  
H. Shin ◽  
Y. Lee ◽  
H. R. Bak ◽  
H. Yu ◽  
...  
Keyword(s):  
Autoimmune Disease ◽  
Autoimmune Diseases ◽  
Whole Blood ◽  
Inflammatory Diseases ◽  
Selective Inhibition ◽  
Janus Kinase ◽  
Dependent Manner ◽  
Human Whole Blood

Background:Janus kinases (JAK1, JAK2, JAK3, and TYK2) play critical roles in mediating various cytokine signaling, and has been developed as a target for autoimmune diseases such as RA. Tofacitinib, oral Pan-JAK inhibitor, demonstrated efficacy in RA patients, but its widespread use is limited by safety issues. Baricitinib, JAK1/2 inhibitor, is also known to interfere with the hematopoiesis system, such as anemia and thrombocytopenia associated with suppression of JAK2 signals. Therefore, it is necessary to develop a new potent compound that selectively inhibits JAK1 over JAK2, 3Objectives:To identify the pharmacological characteristic based on efficacy of CJ-15314 as potent and selective JAK1 inhibitor for treatment of autoimmune disease.Methods:In vitro, cell-based, kinase panel, Kd value and human whole blood assay were performed to determine the inhibition potency and selectivity for JAK subfamily kinases. In vivo therapeutic potential was evaluated by RA model including rat Adjuvant-Induced Arthritis (AIA) and collagen-induced arthritic (CIA). To confirm the possibility of further expansion into the autoimmune disease, BioMAP® Diversity PLUS® Panel was performed by discoverX.Results:In vitro assay, CJ-15314 inhibited JAK kinase family in a concentration-dependent manner with IC50 values of 3.8 nM against JAK1, Selectivity for JAK1 over JAK2, 3 was approximately 18, 83 fold greater for CJ-15314. In 1mM ATP condition, CJ-15314 has been confirmed to have the highest JAK1 selectivity over competing drugs, under 1 mM ATP condition that reflects the physiological environment in the body. Similarly, Kd values has also confirmed the selectivity of JAK1, which is 10 fold higher than JAK2, 3. Accordingly, in human whole blood assays, CJ-15314 is 11 fold more potent against IL-6 induced pSTAT1 inhibition through JAK1 (IC50 value: 70 nM) than GM-CSF-induced pSTAT5 inhibition (JAK2) whereas baricitinib and filgotinib exhibited only 2 fold and 7 fold respectively.In vivo efficacy model, CJ-15314 inhibited disease severity scores in a dose dependent manner. In the rat AIA model, CJ-15314 at 30 mg/kg dose showed 95.3% decrease in arthritis activity score, 51.2% in figotinib at 30 mg/kg, 97.7% showed baricitinib at 10 mg/kg. CJ-15314 showed superior anti-arthritic efficacy than filgotinib. CJ-15314 also minimally affected anemia-related parameters but not bricitinib end of the 2-week treatment. In the rat CIA model, like 10 mg/kg of bricitinib, 30 mg/kg of CJ-15314 also has a similar effect, with a significant reduction in histopathological scores.In biomap diversity panel, CJ-15314 inhibited the expression of genes such as MCP-1, VCAM-1, IP-10, IL-8, IL-1, sTNF-α and HLA-DR confirming the possibility of expansion into other diseases beyond arthritis.Conclusion:CJ-15314 is a highly selective JAK1 inhibitor, demonstrates robust efficacy in RA animal model and is good candidate for further development for inflammatory diseases.* CJ-15314 is currently conducting a phase I trial in south Korea.References:[1]Clark JD et al. Discovery and development of Janus kinase (JAK) inhibitors for inflammatory diseases. J Med Chem. 2014; 57(12):5023-38.[2]Burmester GR et al. Emerging cell and cytokine targets in rheumatoid arthritis. Nat Rev Rheumatol. 2014; 10(2):77-88[3]Jean-Baptiste Telliez et al. Discovery of a JAK3-selective inhibitor: functional differentiation of JAK3-selective inhibition over pan-JAK or JAK1-selective inhibition. ACS Chem. Biol., 2016; 11 (12):3442-3451Disclosure of Interests:so young Ki Employee of: CJ healthcare, hyunwoo shin Employee of: CJ healthcare, yelim lee Employee of: CJ healthcare, Hyoung rok Bak Employee of: CJ healthcare, hana yu Employee of: CJ healthcare, Seung Chan Kim Employee of: CJ healthcare, juhyun lee Employee of: CJ healthcare, donghyun kim Employee of: CJ healthcare, Dong-hyun Ko Employee of: CJ Healthcare, dongkyu kim Employee of: CJ healthcare

In vitro T2 relaxivities of the Gd-based contrast agents (GBCAs) in human blood at 1.5 and 3 T

2018 ◽  
Vol 60 (6) ◽  
pp. 694-701 ◽  
Author(s):  
Yaqi Shen ◽  
Frank L Goerner ◽  
Johannes T Heverhagen ◽  
Christopher Snyder ◽  
Daoyu Hu ◽  
...  
Keyword(s):  
Body Temperature ◽  
Contrast Agents ◽  
Whole Blood ◽  
Medical Literature ◽  
Theoretical Calculations ◽  
Clinical Impact ◽  
Human Whole Blood ◽  
Comprehensive Comparison ◽  
The Relationship

Background The availability of data in the medical literature for the T2 relaxivities of the Gd-based contrast agents (GBCAs) is limited. A comprehensive comparison between the agents available commercially (other than in Europe) is lacking, with no data available that most closely reflect the clinic, which is in human whole blood at body temperature. Purpose To complement the existing literature by determining T2 relaxivity data for eight GBCAs in vitro. Material and Methods The relaxivities of eight GBCAs diluted in human whole blood at 1.5 and 3 T were determined at 37 ± 0.5 °C. Gd was in the range of 0–4 mM. Multi-echo sequences with variable echo times were acquired using a phantom containing a dilution series with each agent, and SigmaPlot 12.0 was used to calculate the R2 relaxation rate and finally r2. Statistical comparisons between agents and field strengths were conducted. Results The relationship between R2 vs. Gd was observed to be linear at 1.5 and 3 T, with a mild increase in r2 from 1.5 to 3 T for all GBCAs. T2 relaxivity data were compared with prior results. The GBCAs are closely clustered into two groups, with higher r2 noted for the two lipophilic (those with partial hepatobiliary excretion) compounds. Conclusion The r2 values at 1.5 and 3 T, determined for the eight GBCAs still clinically available (other than in Europe), provide a definitive baseline for future evaluations, including theoretical calculations of signal intensity and their clinical impact on T2-weighted scans.

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