scholarly journals Ex vivo Immune Profiling in Patient Blood enables Quantification of innate immune Effector Functions

2020 ◽  
Author(s):  
Teresa Lehnert ◽  
Ines Leonhardt ◽  
Sandra Timme ◽  
Daniel Thomas-Rüddel ◽  
Frank Bloos ◽  
...  
Keyword(s):  
Cardiac Surgery ◽  
Whole Blood ◽  
Immune Cell ◽  
Ex Vivo ◽  
Innate Immune ◽  
Response Patterns ◽  
Inflammatory Stimulus ◽  
Functional Parameters ◽  
Immune Profiling ◽  
Biomathematical Modeling

AbstractThe immune response towards infection is a dynamic system combating invading pathogens to maintain homeostasis and the integrity of the body. Unbalanced immune response profiles determine many clinical syndromes including sepsis and thus present a major challenge in management of life threatening infections. Consequently, there is a high demand to determine a patient’s immune status and identifying functional parameters for immune dysfunction.Here, we quantified the global functional status of human innate immune responses by using a human whole-blood model of infection combined with biomathematical modeling. By determining functional parameters of innate immune cell populations after ex vivo whole-blood bacterial (Staphylococcus aureus) and fungal (Candida albicans) infection, we examined cell-specific functional parameters including migration rates or phagocytosis rates in patients that underwent cardiac surgery with extracorporeal circulation. This intervention is known to pose a transient but strong inflammatory stimulus. In addition to a post-operative increase in white blood cell count mainly caused by mobilization of immature neutrophils we find that the surgery induced pro-inflammatory stimulus results in a mitigation of pathogen-specific response patterns that are characteristic for healthy people and baseline results in our patients. Moreover, our model revealed changing rates for pathogen immune evasion, indicating increased inter-pathogenic differences after surgery. This effect was specific for C. albicans and could not be observed for S. aureus. In summary, our model gives insight into immune functionality and might serve as a functional immune assay to record and evaluate innate response patterns towards infection.Author summaryAssessment of a patient’s immune function is critical in many clinical situations. One prominent example is sepsis, which results from a loss of immune homeostasis due to microbial infection. Sepsis is characterized by a plethora of pro- and anti-inflammatory simuli that may occur consecutively or simultaneously and thus any immunomodulatory therapy would require in depth knowledge of an individual patient’s immune status at a given time. Whereas lab-test based immune profiling often relies solely on quantification of cell numbers, we have used an ex vivo whole-blood infection model in combination with biomathematical modeling to quantify functional parameters of innate immune cells in patient blood. A small blood sample of patients undergoing cardiac surgery, which is known to constitute an inflammatory stimulus was infected ex vivo. Functional immune cell parameters were determined using a combination of experimental assays and biomathematical modeling. We show that these parameters change after an inflammatory insult triggered by cardiac surgery and extracorporeal circulation. This does not only interfere with pathogen elimination from blood but also selectively augments the escape of the fungal pathogen Candida albicans from phagocytosis.

scholarly journals Ex vivo immune profiling in patient blood enables quantification of innate immune effector functions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Teresa Lehnert ◽  
Ines Leonhardt ◽  
Sandra Timme ◽  
Daniel Thomas-Rüddel ◽  
Frank Bloos ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Innate Immune ◽  
Infection Model ◽  
Specific Response ◽  
Response Patterns ◽  
Microbial Infection ◽  
Immune Effector ◽  
Inflammatory Stimuli ◽  
Innate Immune Effector ◽  
Immune Profiling

AbstractThe assessment of a patient’s immune function is critical in many clinical situations. In complex clinical immune dysfunction like sepsis, which results from a loss of immune homeostasis due to microbial infection, a plethora of pro- and anti-inflammatory stimuli may occur consecutively or simultaneously. Thus, any immunomodulatory therapy would require in-depth knowledge of an individual patient’s immune status at a given time. Whereas lab-based immune profiling often relies solely on quantification of cell numbers, we used an ex vivo whole-blood infection model in combination with biomathematical modeling to quantify functional parameters of innate immune cells in blood from patients undergoing cardiac surgery. These patients experience a well-characterized inflammatory insult, which results in mitigation of the pathogen-specific response patterns towards Staphylococcus aureus and Candida albicans that are characteristic of healthy people and our patients at baseline. This not only interferes with the elimination of these pathogens from blood, but also selectively augments the escape of C. albicans from phagocytosis. In summary, our model could serve as a valuable functional immune assay for recording and evaluating innate responses to infection.

scholarly journals Ex Vivo Immune Profiling in Patient Blood Enables Quantification of Innate Immune Effector Functions

2021 ◽  
Author(s):  
Teresa Lehnert ◽  
Ines Leonhardt ◽  
Sandra Timme ◽  
Daniel Thomas-Rüddel ◽  
Frank Bloos ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Innate Immune ◽  
Infection Model ◽  
Specific Response ◽  
Response Patterns ◽  
Microbial Infection ◽  
Immune Effector ◽  
Pathogen Elimination ◽  
Inflammatory Stimuli ◽  
Immune Profiling

Abstract Assessment of a patient’s immune function is critical in many clinical situations. One prominent example is sepsis, which results from a loss of immune homeostasis due to microbial infection and is characterized by a plethora of pro- and anti-inflammatory stimuli that may occur consecutively or simultaneously. Thus, any immunomodulatory therapy would require in depth knowledge of an individual patient’s immune status at a given time. Whereas lab-test based immune profiling often relies solely on quantification of cell numbers, we have used an ex vivo whole-blood infection model in combination with biomathematical modeling to quantify functional parameters of innate immune cells in blood from patients undergoing cardiac surgery. These patients experience a well characterized inflammatory insult, which results in mitigation of pathogen-specific response patterns towards Staphylococcus aureus and Candida albicans that are characteristic for healthy people and baseline results in our patients. This does not only interfere with pathogen elimination from blood but also selectively augments the escape of C. albicans from phagocytosis. In summary, our model gives insight into immune functionality and might serve as a functional immune assay to record and evaluate innate response patterns towards infection.

scholarly journals P-MAPA, a Fungi-Derived Immunomodulatory Compound, Induces a Proinflammatory Response in a Human Whole Blood Model

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Mariana Torrente Gonçalves ◽  
Carla Cristina Squaiella-Baptistão ◽  
Giselle Pidde ◽  
Priscila Hess Lopes ◽  
Iseu da Silva Nunes ◽  
...  
Keyword(s):  
Animal Models ◽  
Complement Activation ◽  
Whole Blood ◽  
Cell Activation ◽  
Immune Cell ◽  
Ex Vivo ◽  
Proinflammatory Response ◽  
Human Whole Blood ◽  
Human Leukocytes ◽  
Immunomodulatory Properties

P-MAPA is a complex compound, derived from Aspergillus oryzae cultures, that has shown immunomodulatory properties in infection and cancer animal models. Despite promising results in these models, the mechanisms of cellular activation by P-MAPA, suggested to be Toll-like receptor- (TLR-) dependent, and its effect on human immune cells, remain unclear. Using an ex vivo model of human whole blood, the effects of P-MAPA on complement system activation, production of cytokines, and the expression of complement receptors (CD11b, C5aR, and C3aR), TLR2, TLR4, and the coreceptor CD14 were analyzed in neutrophils and monocytes. P-MAPA induced complement activation in human blood, detected by increased levels of C3a, C5a, and SC5b-9 in plasma. As a consequence, CD11b expression increased and C5aR decreased upon activation, while C3aR expression remained unchanged in leukocytes. TLR2 and TLR4 expressions were not modulated by P-MAPA treatment on neutrophils, but TLR4 expression was reduced in monocytes, while CD14 expression increased in both cell types. P-MAPA also induced the production of TNF-α, IL-8, and IL-12 and oxidative burst, measured by peroxynitrite levels, in human leukocytes. Complement inhibition with compstatin showed that P-MAPA-induced complement activation drives modulation of C5aR, but not of CD11b, suggesting that P-MAPA acts through both complement-dependent and complement-independent mechanisms. Compstatin also significantly reduced the peroxynitrite generation. Altogether, our results show that P-MAPA induced proinflammatory response in human leukocytes, which is partially mediated by complement activation. Our data contribute to elucidate the complement-dependent and complement-independent mechanisms of P-MAPA, which ultimately result in immune cell activation and in its immunomodulatory properties in infection and cancer animal models.

Recombinant Factor VIIa Combined with Fibrinogen Corrects the Coagulopathy Associated with Cardiopulmonary Bypass Surgery Ex Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4097-4097
Author(s):  
Pall T. Onundarson ◽  
Hanna S. Asvaldsdottir ◽  
Brynja R. Gudmundsdottir ◽  
Benny Sorensen
Keyword(s):  
Cardiac Surgery ◽  
Cardiopulmonary Bypass ◽  
Tissue Factor ◽  
Whole Blood ◽  
Ex Vivo ◽  
Maximum Velocity ◽  
Rank Test ◽  
Protamine Sulphate ◽  
Blood Samples ◽  
Increased Risk

Abstract Major cardiac surgery and use of cardiopulmonary bypass (CPB) is often associated with the development of a severe coagulopathy, hyperfibrinolysis and increased risk of bleeding. The present ex vivo study challenged the hypotheses that whole blood thrombelastometry, activated with minute amounts of tissue factor, can reveal the development of a coagulopathy following cardiac surgery, and that supplementation with fibrinogen and rFVIIa, alone or in combination, can improve the ex vivo clotting pattern. In total, 22 patients with a median age of 68, undergoing coronary artery bypass grafting or valve surgery with use of CPB were included in the study. Dynamic thrombelastometric clotting profiles were recorded using citrated whole blood activated with dilute tissue factor (Innovin®, final dilution 1:17000). Blood samples were collected before surgery (control) and immediately following in vivo neutralization of heparin with protamine sulphate. All blood samples for thrombelastometry were treated with heparinase to ensure neutralization of residual heparin. Standard coagulation laboratory parameters and platelet function confirmed the development of a significant coagulopathy following CPB. The post-operative blood samples were spiked with buffer, rFVIIa (2 μg/mL), fibrinogen (1mg/mL), or the combination of rFVIIa+fibrinogen. The post-operative coagulopathy was evident by thromboelastometry as a statistically significant derangements (Wilcoxon signed rank test). There was prolongation of the onset of clotting (CT, from a median value of 183 seconds pre-op to 385 sec post-op), reduced maximum velocity of clot formation (MaxVel, from 17.5 mm*100/sec pre-op to 15.1 post-op) and reduced maximum clot firmness (MCF, from 6234 mm pre-op to 5527 post-op). Ex vivo spiking with rFVIIa shortened the post-operative clot initiation phase (CT) to a median of 232 sec. Fibrinogen also shortened the post-operative clotting time to a median of 246 sec, and additionally increased the MCF to 5839 mm. Finally, the combination of rFVIIa and fibrinogen together corrected the abnormal thromboelastometric findings associated with CPB-coagulopathy into the pre-operative range, i.e. median CT decreased to 155 sec, MaxVel increased to 16.8 mm*100/sec and MCF increased to 5808 mm. In conclusion, the experiments suggest a potential role of fibrinogen supplementation during control of bleeding following CPB, either alone or in combination with rFVIIa, since the combination corrected the CPB-associated coagulopathy remaining following neutralization of heparin.

scholarly journals Tranexamic acid modulates the immune response and reduces postsurgical infection rates

2019 ◽  
Vol 3 (10) ◽  
pp. 1598-1609 ◽  
Author(s):  
Dominik F. Draxler ◽  
Kah Yep ◽  
Gryselda Hanafi ◽  
Anoushka Winton ◽  
Maria Daglas ◽  
...  
Keyword(s):  
Cardiac Surgery ◽  
Immune System ◽  
Tranexamic Acid ◽  
Postoperative Infection ◽  
Immune Cell ◽  
Innate Immune ◽  
Innate Immune Cell ◽  
Independent Effect ◽  
Surgical Trauma ◽  
Infection Rates

Abstract Tranexamic acid (TXA) is an antifibrinolytic agent that blocks plasmin formation. Because plasmin is known to promote inflammatory and immunosuppressive responses, we explored the possibility that plasmin-mediated immunosuppression in patients undergoing cardiac surgery can be directly reversed by TXA and decrease postoperative infection rates. The modulatory effect of TXA on inflammatory cytokine levels and on innate immune cell activation were evaluated with multiplex enzyme-linked immunosorbent assay and flow cytometry, respectively. Postoperative infection rates were determined in patients undergoing cardiac surgery and randomized to TXA (ACTRN12605000557639; http://www.anzca.edu.au). We demonstrate that TXA-mediated plasmin blockade modulates the immune system and reduces surgery-induced immunosuppression in patients following cardiac surgery. TXA enhanced the expression of immune-activating markers while reducing the expression of immunosuppressive markers on multiple myeloid and lymphoid cell populations in peripheral blood. TXA administration significantly reduced postoperative infection rates, despite the fact that patients were being administered prophylactic antibiotics. This effect was independent of the effect of TXA at reducing blood loss. TXA was also shown to exert an immune-modulatory effect in healthy volunteers, further supporting the fibrin-independent effect of TXA on immune function and indicating that baseline plasmin levels contribute to the regulation of the immune system in the absence of any comorbidity or surgical trauma. Finally, the capacity of TXA to reduce infection rates, modulate the innate immune cell profile, and generate an antifibrinolytic effect overall was markedly reduced in patients with diabetes, demonstrating for the first time that the diabetic condition renders patients partially refractory to TXA.

Abstract P149: Germ-free Rats Reveal An Obligatory Role Of Microbiota In Blood Pressure

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Saroj CHAKRABORTY ◽  
Xi Cheng ◽  
Cam McCarthy ◽  
Blair Mell ◽  
Ji-youn Yeo ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Vascular Function ◽  
Immune Cell ◽  
Ex Vivo ◽  
Innate Immune ◽  
Functional Evaluation ◽  
Immune Cell Population ◽  
16S Sequencing ◽  
Germ Free ◽  
Vascular Contractility

Elevated blood pressure or hypertension is the single largest risk factor for cardiovascular diseases which are the leading cause of human deaths. Current clinical management of blood pressure is focused on restoring homeostasis of the host alone, without accounting for commensal gut microbiota. Recent evidence from the CARDIA study in humans and multiple studies using animal models suggest that development of hypertension in the host is associated with alterations in microbiotal communities. Here we examined whether microbiota is necessary for blood pressure and vascular homeostasis by functional evaluation of the gut homeostasis, hemodynamic, and vascular function of gnotobiotic rats reconstituted with microbiota to represent the complete holobiont. Gnotobiotic rats were used to represent incomplete holobionts. To reconstitute complete holobionts, gnotobiotic rats were co-housed with conventionally-raised rats. Acquisition of microbiota was evaluated through monitoring of gross ceca and fecal samples by metagenomic 16S sequencing. BP was recorded and vascular, renal, hepatic, cardiac and gut features were assessed using histology and ex vivo myography. Markers of innate immune effectors (Immune cell population, level of Lcn2, Gut permeability) were used to examine the nature and extent of host immune cell processes concomitantly occurring along with observations of host hemodynamics. Compared to the reconstituted holobiont represented by the animals exposed to microbiota, the incomplete-holobiont represented by gnotobiotic rats, had significantly lower BP (SBP of germ free:109±8 mmHg, SBP of conventionalized:138±10mmHg * ) and vascular contractility responses to phenylephrine (Emax (mN): germ-free: 6.9±1.3, GFC: 11.7±0.7*). Acute exposure of the host to microbiota reconstituted gut microbiotal communities, significantly boosted their gut epithelial cell proliferation, innate immune function and restored vascular contractility. These data indicate that in addition to the dependency of the host on microbiota for essential bodily functions such as digestion of plant-derived complex carbohydrates, the host is also dependent on microbiota for maintaining blood pressure and vascular function

scholarly journals Donor-Specific Plasma Factors Contribute to Aberrant Staining Artifacts with a Commercial Lyophilized Mass Cytometry Antibody Panel

2020 ◽  
Author(s):  
Daniel Geanon ◽  
Brian Lee ◽  
Adeeb H. Rahman
Keyword(s):  
Whole Blood ◽  
Immune Cell ◽  
Immune Monitoring ◽  
Mass Cytometry ◽  
Specific Antibodies ◽  
Design And Optimization ◽  
Wide Range ◽  
Antibody Panel ◽  
Plasma Factors ◽  
Immune Profiling

AbstractHigh-dimensional mass cytometry (CyTOF) phenotyping allows for the routine measurement of over 40 parameters and is increasingly being utilized across a wide range of studies. However, CyTOF-specific panel design and optimization represent challenges to wider adoption and standardization of immune profiling with CyTOF. To address this, Fluidigm recently commercialized its MaxPar Direct Immune Profiling Assay (MDIPA), which comprises a lyophilized 30-marker antibody panel that is able to identify all major circulating immune cell subsets and offers a streamlined solution for standardized human immune monitoring. However, in the course of applying the MDIPA to characterize large numbers of whole blood samples, we observed several instances of unusual aberrant staining patterns, most notably CD19 expression on non-B cells, which can potentially confound data analysis and lead to erroneous interpretation of results when using this assay. Here, we report that this complex phenomenon is mediated by donor-specific plasma factors that mediate non-specific interactions between specific antibodies in the MDIPA panel. Our findings additionally suggest specific strategies that can be used to mitigate the issue, including the use of PBMCs or lysed/washed whole blood to remove endogenous plasma prior to staining, or blocking specific antibodies in the MDIPA panel.

scholarly journals Increased TNF-α Initiates Cytoplasmic Vacuolization in Whole Blood Coculture with Dengue Virus

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Rahmat Dani Satria ◽  
Tzu-Wen Huang ◽  
Ming-Kai Jhan ◽  
Ting-Jing Shen ◽  
Po-Chun Tseng ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Whole Blood ◽  
Ex Vivo ◽  
Nonstructural Protein ◽  
Denv Infection ◽  
Cytopathic Effects ◽  
Cytoplasmic Vacuolization ◽  
Tnf Α ◽  
A Minor ◽  
Immune Profiling

During the acute febrile phase of dengue virus (DENV) infection, viremia can cause severe systemic immune responses accompanied by hematologic disorders. This study investigated the potential induction and mechanism of the cytopathic effects of DENV on peripheral blood cells ex vivo. At one day postinfection, there was viral nonstructural protein NS1 but no further virus replication measured in the whole blood culture. Notably, DENV exposure caused significant vacuolization in monocytic phagocytes. With a minor change in the complete blood cell count, except for a minor increase in neutrophils and a significant decrease in monocytes, the immune profiling assay identified several changes, particularly a significant reduction in CD14-positive monocytes as well as CD11c-positive dendritic cells. Abnormal production of TNF-α was highly associated with the induction of vacuolization. Manipulating TNF-α expression resulted in cytopathogenic effects. These results demonstrate the potential hematological damage caused by ex vivo DENV-induced TNF-α.

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