A Human Whole Blood Culture System Reveals Detailed Cytokine Release Profiles of Implant Materials.

Introduction: Common in vitro cell culture systems for testing implant material immune compatibility either rely on immortal human leukocyte cell lines or isolated primary cells. Compared to in vivo conditions, this generates an environment of substantially reduced complexity, often lacking important immune cell types, such as neutrophil granulocytes and others. The aim of this study was to establish a reliable test system for in vitro testing of implant materials under in vivo-like conditions. Methods: Test materials were incubated in closed, CO2-independent, tube-based culture vessels containing a proprietary cell culture medium and human whole blood in either a static or occasionally rotating system. Multiplex cytokine analysis was used to analyze immune cell reactions. Results: To demonstrate the applicability of the test system to implant materials, three commercially available barrier membranes (polytetrafluoroethylene (PTFE), polycaprolactone (PCL) and collagen) used for dental, trauma and maxillofacial surgery, were investigated for their potential interactions with immune cells. The results showed characteristic differences between the static and rotated incubation methods and in the overall activity profiles with very low immune cell responses to PTFE, intermediate ones to collagen and strong reactions to PCL. Conclusion: This in vitro human whole blood model, using a complex organotypic matrix, is an excellent, easily standardized tool for categorizing immune cell responses to implant materials. Compared to in vitro cell culture systems used for materials research, this new assay system provides a far more detailed picture of response patterns the immune system can develop when interacting with different types of materials and surfaces.

Systematic Investigation of Polyurethane Biomaterial Surface Roughness on Human Immune Responses in vitro.

It has been widely shown that biomaterial surface topography can modulate host immune response, but a fundamental understanding of how different topographies contribute to pro-inflammatory or anti-inflammatory responses is still lacking. To investigate the impact of surface topography on immune response, we undertook a systematic approach by analyzing immune response to eight grades of medical grade polyurethane of increasing surface roughness in three in vitro models of the human immune system. Polyurethane specimens were produced with defined roughness values by injection molding according to the VDI 3400 industrial standard. Specimens ranged from 0.1 µm to 18 µm in average roughness (Ra), which was confirmed by confocal scanning microscopy. Immunological responses were assessed with THP-1-derived macrophages, human peripheral blood mononuclear cells (PBMCs), and whole blood following culture on polyurethane specimens. As shown by the release of pro-inflammatory and anti-inflammatory cytokines in all three models, a mild immune response to polyurethane was observed, however, this was not associated with the degree of surface roughness. Likewise, the cell morphology (cell spreading, circularity, and elongation) in THP-1-derived macrophages and the expression of CD molecules in the PBMC model on T cells (HLA-DR and CD16), NK cells (HLA-DR), and monocytes (HLA-DR, CD16, CD86, and CD163) showed no influence of surface roughness. In summary, this study shows that modifying surface roughness in the micrometer range on polyurethane has no impact on the pro-inflammatory immune response. Therefore, we propose that such modifications do not affect the immunocompatibility of polyurethane, thereby supporting the notion of polyurethane as a biocompatible material.

Standardized whole blood stimulation improves immunomonitoring of induced immune responses in multi-center study.

Functional immune responses are increasingly important for clinical studies, providing in depth biomarker information to assess immunotherapy or vaccination. Incorporating functional immune assays into routine clinical practice has remained limited due to challenges in standardizing sample preparation. We recently described the use of a whole blood syringe-based system, TruCulture®, which permits point-of-care standardized immune stimulation. Here, we report on a multi-center clinical study in seven FOCIS Centers of Excellence to directly compare TruCulture to conventional PBMC methods. Whole blood and PBMCs from healthy donors were exposed to LPS, anti-CD3 anti-CD28 antibodies, or media alone. 55 protein analytes were analyzed centrally by Luminex multi-analyte profiling in a CLIA-certified laboratory. TruCulture responses showed greater reproducibility and improved the statistical power for monitoring differential immune response activation. The use of TruCulture addresses a major unmet need through a robust and flexible method for immunomonitoring that can be reproducibly applied in multi-center clinical studies. ONE SENTENCE SUMMARY: A multi-center study revealed greater reproducibility from whole blood stimulation systems as compared to PBMC stimulation for studying induced immune responses.

Functional analysis via standardized whole-blood stimulation systems defines the boundaries of a healthy immune response to complex stimuli.

Standardization of immunophenotyping procedures has become a high priority. We have developed a suite of whole-blood, syringe-based assay systems that can be used to reproducibly assess induced innate or adaptive immune responses. By eliminating preanalytical errors associated with immune monitoring, we have defined the protein signatures induced by (1) medically relevant bacteria, fungi, and viruses; (2) agonists specific for defined host sensors; (3) clinically employed cytokines; and (4) activators of T cell immunity. Our results provide an initial assessment of healthy donor reference values for induced cytokines and chemokines and we report the failure to release interleukin-1α as a common immunological phenotype. The observed naturally occurring variation of the immune response may help to explain differential susceptibility to disease or response to therapeutic intervention. The implementation of a general solution for assessment of functional immune responses will help support harmonization of clinical studies and data sharing.

Identification of a molecular profile associated with immune status in first-onset schizophrenia patients.

OBJECTIVES: Alterations in immunological parameters have been reported for schizophrenia although little is known about the effects of inflammatory status on immune-related functional changes at disease onset. Here, we have investigated such T cell-dependent molecular changes in first-onset, antipsychotic-naive schizophrenia patients using a novel ex vivo blood culture system. METHODS: Blood samples from patients (n=17) and controls (n=17) were collected into stimulant-containing or null control TruCulture™ tubes, incubated 24 hours and the concentrations of 107 immune and metabolic molecules measured in the conditioned media using the HumanMAP™ immunoassay system. RESULTS: Nine molecules showed altered release from schizophrenia blood cells compared to those from controls and this was replicated in an independent cohort. In silico pathway analysis showed that these molecules had roles in endothelial cell function, inflammation, acute phase response and fibrinolysis pathways. Importantly, five of these molecules showed altered release only after stimulation. CONCLUSIONS: This study has identified a reproducible peripheral molecular signature associated with altered immune function in first-onset schizophrenia subjects. This suggests that immune status can affect the biomarker profile which could be important for personalized medicine strategies. Furthermore, whole blood culture analysis may be useful in the identification of diagnostic tools or novel treatment strategies due to ease-of-use and clinical accessibility.

Placebo-controlled randomized clinical trial on the immunomodulating activities of low- and high-dose bromelain after oral administration - new evidence on the antiinflammatory mode of action of bromelain.

Bromelain has been used for treatment of inflammatory diseases for decades. However, the exact mechanism of action remains poorly understood. While in vitro investigations have shown conflicting effects on the release of various cytokines, no in vivo data were available. In this study, the effects on inflammation-related cytokines of two doses of bromelain were tested in a single dose placebo-controlled 3 × crossover randomized clinical trial. Cytokine circadian profiles were used to investigate the effects of bromelain on the human immune system by using stimulated whole-blood leukocytes. The effects seen in these cultures demonstrated a significant shift in the circadian profiles of the Th1 cell mediator interferon gamma (IFNγ; p < 0.043) after bromelain 3000 FIP (Fédération Internationale Pharmaceutique) units, and trends in those of the Th2-type cytokine IL-5 as well as the immunosuppressive cytokine interleukin (IL)-10. This suggests a general effect on the antigen-specific (T cell) compartment of the human immune system. This is the first time that bromelain has been shown to modulate the cellular responses of lymphocyte after oral use. It is postulated that the immunomodulating effect of bromelain observed in this trial is part of its known antiinflammatory activities. Further investigations will be necessary to verify the relevance of these findings to a diseased immune system.

Characterization of immunologically active drugs in a novel organotypic co-culture model of the human gut and whole blood.

The human immune system represents a highly complex multicellular network that protects the organism against the environment and pathogens. Within this system, different immune cells communicate with each other, as well as with adjacent organs and tissues, using an impressive network of regulatory signals. This inherent complexity makes it rather difficult to mimic these processes in vitro. Unpredictable drug-induced side effects can be the consequence when moving from preclinical animal models into clinical phase. Therefore, there is a demand for more elaborate in vivo like human cell culture models. In this study, an in vitro co-culture model consisting of Caco-2 human gut epithelial cells and human whole blood representing the immune system is applied to investigate the intestinal absorption of anti-inflammatory drugs and the subsequent modulation of the immunoregulatory signaling processes. By using blood of different donors, the individuality of the immune system is integrated into the overall analysis. The anti-inflammatory drugs prednisolone and ibuprofen were applied on top of the Caco-2 epithelial cells and alterations in the extracellular communication via cytokines and chemokines were visualized using miniaturized multiplexed sandwich immunoassays. Optionally, pretreatment of the Caco-2 epithelial cells with pro-inflammatory mediators can be used to modulate the epithelial barrier function similar to the situation observed during inflammatory conditions of the gut. The presented translational test system, consisting of differentiated Caco-2 intestinal epithelial cells and whole blood substantially improves preclinical screening of immunologically active drugs with respect to an approximation of the human "in vivo" conditions.

Intraindividual long term stability and response corridors of cytokines in healthy volunteers detected by a standardized whole-blood culture system for bed-side application.

BACKGROUND: The variation of immune cell activities over time is an immanent property of the human immune system, as can be measured by the stimulated secretion of cytokines in cell cultures. However, inter-individual variability is considerably higher. Especially the latter is the major reason why it has not been possible to establish international standard values for cytokines as was possible for other parameters, such as leukocyte sub-population numbers. In this trial, a highly standardized whole-blood culture model (TrueCulture®), developed to characterise drug effects on cells of the human immune system in clinical trials, was used to analyse cytokine patterns in the blood samples of 12 healthy subjects over a period of one month. METHODS: After an overnight fast, 12 healthy subjects donated blood three times a week on three consecutive days over a period of 4 weeks. TruCulture® blood collection and whole-blood culture systems were used to measure whole-blood leukocyte stimulation. The levels of IL-2, IL-5, IL-13, IL-6, IL-8, IL-10, IFNγ, and MCP-1 in the culture supernatants were quantified by sandwich ELISA. RESULTS: The pattern of cytokine concentrations in the supernatants of the stimulated whole-blood cultures was highly individual, but considerably stable over the whole observation period of 4 weeks. CONCLUSIONS: By using TruCulture® it seems feasible to determine subject-specific cytokine reference patterns, for example under healthy conditions, or before starting an experimental treatment, e.g. during a clinical trial, against which changes in the behaviour of the immune system can be detected more accurately in future.

Phagocytosis and digestion of pH-sensitive fluorescent dye (Eos-FP) transfected E. coli in whole blood assays from patients with severe sepsis and septic shock.

The function of phagocytic and antigen presenting cells is of crucial importance to sustain immune competence against infectious agents as well as malignancies. We here describe a reproducible procedure for the quantification of phagocytosis by leukocytes in whole blood. For this, a pH-sensitive green-fluorescent protein- (GFP) like dye (Eos-FP) is transfected into infectious microroganisms. After UV-irradiation, the transfected bacteria emit green (≈5160 nm) and red (≈581 nm) fluorescent light at 490 nm excitation. Since the red fluorescent light is sensitive to acidic pH, the phagocytosed bacteria stop emitting red fluorescent light as soon as the phagosomes fuse with lysosomes. The green fluorescence is maintained in the phagolysosome until pathogen degradation is completed. Fluorescence emission can be followed by flow cytometry with filter settings documenting fluorescence 1 (FL 1, FITC) and fluorescence 2 (FL 2, phycoerythrin, PE). Eos-FP transfected bacteria can also be traced within phagocytes using microscopical techniques. A standardized assay has been developed which is suitable for clinical studies by providing clinicians with syringes pre-filled with fixed and appropriately UV-irradiated Eos-FP E. coli (TruCulture™). After adding blood or body fluids to these containers and starting the incubation at 37°C, phagocytosis by granulocytes proceeds over time. Cultures can be terminated at a given time by lysing red blood cells followed by flow cytometry. A pilot study demonstrated that Eos-FP E. coli phagocytosis and digestion was up-regulated in the majority of patients with either severe sepsis or septic shock as compared to healthy donors (p < 0.0001 after o/n incubation). Following treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in selected patients with sepsis, phagolysosome fusion appeared to be accelerated.

An Innovative, Centrifugation-free Method to Prepare Human Platelet Mediator Concentrates Showing Activities Comparable to Platelet-rich Plasma .

UNLABELLED: Slow-healing wounds benefit considerably from treatment with platelet-rich plasma (PrP). The drawback of using PrP is its laborious preparation, which requires expensive technical equipment (centrifuges) and well-trained personnel. METHODS: The authors' new method overcomes these issues and provides the practitioner with an innovative tool to freshly prepare a platelet mediator mix with PrP's known biological activities, but is much simpler to obtain. This is achieved by employing the sedimentation of a blood sample at regular gravity (no centrifuge necessary) in the presence of an anti-coagulant and a sedimentation accelerator. Thereafter, the supernatant containing the platelets is concentrated on a unique filter, which causes these platelets to release their mediators (different biologically active molecules resembling the substance mix that is released by the platelets upon degranulation). This solution is eluted from the filter, providing a sterile-filtered, enriched fraction of biologically active mediators (TGF-ß, PDGF, IGF-1, etc.), most of which are active in wound healing disorders. RESULTS: This preparation triggers in-vitro proliferation of fibroblasts and osteoblasts, the secretion of IL-6 by osteoblasts, and differentiation of fibroblasts into cells with an endothelial morphology resembling cells during angiogenesis. CONCLUSION: By providing the practioner a sterile concentrate of a whole range of autologous platelet mediators within 1 hour, this new method has the potency to become a substitute of PrP in wound-healing therapy. PMC (platelet mediator concentrate) eases the manufacturing of such preparations, thereby making them not only more widely applicable, but also reducing treatment costs.

Functional drug candidate profiling using complex human organotypic cell culture models: a promising way to reduce clinical drug failure.

The human immune system is one of the most important causative factors in the pathogenesis of various chronic inflammatory diseases. The network of immunoregulatory signals ruling the course of such disorders is highly complex and does not only involve the cells of the immune system, but also those of the diseased organs. This makes it rather difficult to model such regulatory processes in vitro. Most existing cell culture systems suffer from a serious lack of complexity. This is one of the major reasons why the majority of drug candidates fail in patients. Drugs that are meant to mono-specifically counter-regulate a disease process tend to develop unpredictable responses, due to unknown crosslinks in signalling pathways that are not available in simple culture models. Thus, the candidate selection as it is used at present, can be improved substantially by using more elaborate, in-vivo-like human cell culture models. Such test systems will accelerate the drug development process and decrease the risk of candidate failure. This review describes a new set of human organotypic, reproducible routine cell culture models, developed to optimise the drug candidate selection process and to increase clinical success rates. It is expected that models like these will accelerate drug development in the future substantially.