Immune Responsiveness and Outcome After Aortic Valve Surgery (Measure)

Summary

Participation Requirements

Description

Experimental design and methods: Setting: Barts Heart Centre is a 255-bedded specialist cardiac centre where approximately 200 primary isolated open aortic valve replacement procedures are performed per year. It is closely associated both geographically and academically with the William Harvey Resea...

Experimental design and methods: Setting: Barts Heart Centre is a 255-bedded specialist cardiac centre where approximately 200 primary isolated open aortic valve replacement procedures are performed per year. It is closely associated both geographically and academically with the William Harvey Research Institute (WHRI), which forms part of Queen Mary University, London (QMUL). This will allow laboratory analysis of all collected blood samples within 15 minutes if necessary. Barts Heart Centre is one of the largest cardiac units in Europe and a key research objective of QMUL/WHRI is to support cardiac research. Sample collection and storage: Following written informed consent all patients will be pre-operatively risk-scored by means of ASA, Euroscore 2 and conventionally risk-scored for the development of surgical site infection. Blood (44ml total) will be taken from 150 aortic valve replacement patients immediately prior to surgery (T0). Blood (7ml) will be collected into a PAXgene tube and stored at -80° for later genetic analysis. A clotted sample (7ml) will be collected in a standard gold top SST tube, left to clot then centrifuged for 10 mins at 1000g, the serum separated and stored at -80° for later analysis of antibody levels. In addition, 30 ml blood will be taken for study of peripheral blood mononuclear cells (PBMCs), drawn into standard purple top EDTA tubes (anticoagulant). These will be separated within 3 hours of collection by Ficoll-Paque density gradient centrifugation, whereby PBS-diluted blood is carefully layered over Ficoll and spun brake off in a centrifuge at 400g for 30 minutes. The PBMC layer is then extracted and washed twice in PBS and stored in 10% dimethyl sulfoxide freezing solution in liquid nitrogen so that they can be thawed, and batch analysed at a later date. Sample analysis: PBMCs will be counted using a haemocytometer, then cultured in RPMI and 10% serum with either LPS, ?-toxin or unstimulated for 24 hours, after which they will be analysed immediately as detailed below. • Soluble Mediators (Plasma) Storage: Ethylenediaminetetraacetic acid (EDTA, BD Biosciences Vacutainer, 9mL) anticoagulated blood will be centrifuged (1200g, 10mins, 20°C) and plasma stored at -80°C within 2hrs of collection Analysis: Cytokines: Meso Scale Discovery (MSD) V-PLEX Proinflammatory Panel 1 will be employed to quantify IFN-?, IL-10, IL-12p70, IL-13, IL-1?, IL-2, IL-4, IL-6, IL-8, and TNF-?. Plates will be read using a MSD QuickPlex SQ 120 imager (Wiliam Harvey Institute, QMUL). Antibodies: These will be measured by means of ELISA assays against staphylococcal antigens (?-toxin, teichoic acid) and core moieties of the endotoxin molecule (EndoCAb). Whole blood LPS/?-toxin Stimulated Cytokine Release Technique: As previously described and validated in our laboratory, heparinized blood will be stimulated for 4hrs (37°C, 250rpm) with 1ng/ml LPS within 2hrs of draw. After incubation, samples will be centrifuged (1200g, 10mins, 20°C) and supernatant stored at -80°C. Cytokine quantification: As per 'soluble mediators' MSD V-PLEX Proinflammatory Panel 1 will be employed to quantify TNF-? (primary outcome as metric of immune competence) and other cytokines. Cytokines will be expressed as a factor of the number of circulating monocytes. Flow Cytometric Immunophenotyping and Functional Assessment of Leukocytes Flow Cytometry: All samples will be analysed on the same machine (Becton Dickinson (BD) Fortessa, Rayne Building, UCL). Standardisation and comparison of output will be achieved via employment of constant voltages and compensation matrix throughout with daily matching of values to a single batch of Cytometry Setup and Tracking (CS&T) beads (BD). Leukocyte cell surface staining will be conducted using antibodies principally supplied by BD. Staining, data capture and storage will be conducted in accordance with a single study standard operating procedure. 5 panels (up to 14-colour) and two functional assays will be performed at each time-point. Data will be analysed in FlowJo version 10.5. Surface Marker Staining: Quantification and calibration panel: CD45, CD56, CD3, CD4, CD8, CD19, CD14, CD16, performed in a BD TruCount tube to enable enumeration of cell subsets Cell specific panels (incorporating markers of sub-categorisation and markers of immune competence) Monocyte/Myeloid: CD3, CD19, CD56, CD163, CD16, CD33, CD141, CD206, CD274, CD14, CD1c, CD11c, HLA-DR, CD80, CD123, CD83 Neutrophil: CD3, CD19, CD56, HLA-DR, CD64, CD62L, CD11b, CD88, CD66b, CD14, CD11c, CD16, CD184, CD182 Lymphocyte: CD19, CCR6, CD45RA, CD3, CD4, CD8, CXCR3, CD127, CD62L, CD25, CD56, CD27, CD279 HLA-DR Expression: Will be quantified on CD14+ monocytes using BD QuantiBrite beads. Functional Measures Phagocytosis: The ability of neutrophils and monocytes to phagocytose opsonised FITC-labelled E.coli bacteria will be quantified via the PhagoTest assay (BD) as per manufacturer's instructions Reactive oxygen 'burst': Quantitative determination of leukocyte oxidative burst will be undertaken using the PhagoBurst assay (BD) in response to un-labelled opsonized E.coli, phorbol 12-myristate 13-acetate (PMA) and the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP). o The pre-surgery sample PBMCs that will be stimulated with LPS (type and concentration to be optimized via dose response curve on healthy volunteers) and ?-toxin will undergo flow cytometry to measure TLR4 and TLR5 surface expression, HLA-DR surface expression and quantification of NF-kB using appropriate antibodies validated in the literature. At the end of stimulation, cells will be harvested, and RNA extracted for quantitative (q)PCR to evaluate AID mRNA expression. Although B cells in the PBMC cultures have been stimulated in the presence of other cell types, primarily T cells and monocytes-macrophages, our endpoint is to measure a B-cell response, as AID is exclusively expressed in B cells. Enough healthy volunteer blood is available so that all above techniques can be adequately practiced and optimized prior to patient recruitment. Further sampling timepoints will be immediately post-op (T1), 24h post-op (T2), discharge (T3) and 4 weeks post-op at surgical follow up (T4). The blood collected at 4-week post-surgery follow up will be assayed for IgG antibody assay to EndoCAb, ?-toxin and teichoic acid. This will enable a measure of class switch recombination. Which will be referable back to the degree of expression of AID mRNA. The population of aortic valve replacement patients will be followed up for mortality and major morbidity. They will be scored as per C-POMS (cardiac post-operative morbidity score), length of ITU stay and length of hospital stay. Patients will be monitored for development of surgical site infection (SSI) or the acquisition of post-operative hospital acquired infection (HAI). Post-discharge follow-up for wound infection or breakdown will be continued for 12 months. At the immediately pre-operative timepoint (T0), alongside currently accepted measures of immune responsiveness it is intended to perform a near patient test of immune responsiveness (LIT) using a handheld chemiluminescence device that relies upon leukocyte oxidative burst to a supramaximal stimulus (Oxford Medistress). This relies upon analysis of the response in a 10mcl fresh capillary blood sample. Statistical considerations: From published data from centres worldwide it is known that a proportion of patients develop an infection, either surgical site infection (SSI) or hospital acquired infection (HAI), following aortic valve surgery. It is also known that these patients endure longer stay in hospital following surgery. The investigators have demonstrated that amongst these patients there is variability in levels of antibodies to various perioperative threats, namely endotoxin and staphylococcus. The investigators have demonstrated that in the patients who are undergoing surgery and have low levels of circulating antibody to endotoxin and/or staphylococcus there is an association with greater likelihood of developing an infection and a longer length of post-operative stay. The investigators estimate that the rate of infection varies between 13.5 - 33%. The investigators have previously suggested that the association between antibody level and outcome may be manifestation of an underlying impaired ability in some individuals to mount a normal immune response to these threats. It is known that a proportion of individuals have an abnormal immune response. This is perhaps most obviously seen in the response to vaccination where previous work has demonstrated the lack of immune responsiveness following various vaccination programs and our own work on EndoCAb response to monovalent typhoid vaccination. This non/poor responsiveness in these studies lies somewhere between 10 and 40%. The diagram illustrates the proportion of non-responders (ie poor immune responsiveness) if they are equally distributed.

Tracking Information