Sterile post-traumatic immunosuppression.

After major trauma, the human immune system initiates a series of inflammatory events at the injury site that is later followed by suppression of local inflammation favoring the repair and remodeling of the damaged tissues. This local immune response involves complex interactions between resident cells such as macrophages and dendritic cells, soluble mediators such as cytokines and chemokines, and recruited cells such as neutrophils, monocytes and mesenchymal stromal cells. If of sufficient magnitude, these initial immune responses nevertheless have systemic consequences resulting in a state called post-traumatic immunosuppression (PTI). However, controversy exists regarding the exact immunological changes occurring in systemic compartments triggered by these local immune responses. PTI is one of the leading causes of post-surgical mortality and makes patients vulnerable to hospital-acquired infections, multiple organ failure and many other complications. In addition, hemorrhage, blood transfusion, immunesenescence and immunosuppressant drugs aggravate PTI. PTI has been intensively studied, but published results are frequently cloudy. The purpose of this review is to focus on the contributions made by different responsive modalities to immunosuppression following sterile trauma and to try to integrate these into an overall scheme of PTI.

The correlation between the percent of CD3- CD56+ cells and NK precursor function.

The number and function of human natural killer (NK) cells are generally assessed to monitor the baseline of immune function, the effect of treatment, the progress of malignancy or metastases and diseases. NK cells recognise and kill target cells in the absence of prior sensitisation and are able to defend the host from infection or prevent the progression of a disease. Human NK cells express CD16 and CD56 which are (massively) being used as a major hallmark for the NK cell. The purpose of this study was to identify the unique subsets of peripheral blood mononuclear cells (PBMC) (%CD3-CD56+ cells) by flow cytometry and to determine whether there is any correlation with functionally mature progeny of (NKp) precursor after five days of culture. The correlation was analysed using samples obtained from 120 Caucasian patients. 20-30ml of whole blood was collected in sterile tube containing preservative free sodium heparin and a similar sample was obtained after five days. Maturation of NKp required the continuous presence of recombinant interleukin 2 (rIL-2), or interleukin 15 (rIL-15) and functional maturity of NK cells was determined by their ability to lyse target cells from the K562 cell line. The NK precursor frequency was measured by limiting dilution analysis (LDA), which The NKpf assay was set up with a range of cell dilutions from 40,000 to 625 per 100ml/well in 96 well culture plates. At the end of the culture period the K562 cell line labelled with Europium (Eu-K562) was added and Eu release measured in culture supernatants using time-resolved fluorometry. The PBMC were set up in parallel cultures under various conditions . On day five cells were collected from culture plates and adjusted to 1x10 cells/ml and then mixed. The mixture was incubated and anti CD3 and anti CD56 were added. NK cells were enumerated in 120 patients by double staining with a combination of anti-CD3- and anti-CD56+. The results of these Immunophenotyping studies by flow cytometry showed no correlation between the NKpf (natural killer precursor frequency) and the percent of CD3-CD56+ cells expressed after five days confirming that CD56 was inadequate as a unique marker for functional NK cells.

Prognostic assays for rejection and tolerance in organ transplantation.

In this review, I have summarised our understanding of acute rejection of organ transplants, and for convenience I have identified three processes, recognition, rejection and regulation. In stark contrast to this text-book picture of acute rejection, I have drawn attention to some of the clinical realities, where processes are altered by powerful immunosuppressive drugs, and where many transplant recipients are pre-sensitised to transplantation antigens prior to engraftment. The ultimate goal is to encourage the emergence of a utopian immunological state, wherein patients tolerate organ transplants for life after being weaned from all immunosuppressive drugs. Assays that may be used in the future to reliably monitor this process are still at a very exciting stage of development.

Effect of autologous salvaged blood on postoperative natural killer cell precursor frequency.

BACKGROUND: Immunosuppression after major surgery increases the risk of infections. Natural killer cells play a pivotal part in defence against infection. We aimed to investigate the immunomodulatory effects of different types of postoperative blood transfusion by use of a new assay for measuring the frequency of peripheral blood natural killer precursor cells (NKpf assay). METHODS: We measured the natural killer cell precursor (NKp) frequency before and 5 days after surgery in 120 patients undergoing joint replacement surgery. The patients were assigned to one of five groups according to the type of transfusion received: non-transfused (n=32), allogeneic non-leukodepleted blood (eight), allogeneic leukodepleted blood (30), autologous predeposited blood (ten), and autologous salvaged blood collected within the first 24 h after surgery (40). We also measured interferon gamma and interleukin 10 concentrations before and after surgery. FINDINGS: The mean postoperative NKp frequency for all patients was lower than the preoperative values, except in patients receiving autologous salvaged blood, which was higher than all other groups (p<0.0001). Postoperative NKp frequencies for patients receiving allogeneic or autologous predeposited blood responded similarly (p=0.99), but these patients had lower NKp frequencies than did the non-transfused group (p<0.0001). Postoperative interferon gamma concentrations were higher in the autologous salvaged blood group (p<0.0001) than in other groups, which did not differ from each other. Interleukin 10 concentrations were similar across all groups (p=0.49). INTERPRETATION: Immunosuppression associated with surgery and blood loss was reflected in a reduced frequency of NKp and decreased interferon gamma. This immunosuppression was reversed by transfusion of autologous salvaged blood, suggesting that this fluid contained immunostimulants.

Interleukin-10 and tumor necrosis factor alpha region haplotypes predict transplant-related mortality after unrelated donor stem cell transplantation.

Certain cytokine gene polymorphisms have been shown to correlate with outcome of human leukocyte antigen (HLA) identical sibling donor stem cell transplantation (SCT), but in unrelated donor SCT such information is scarce. We have studied the association between cytokine gene polymorphism and transplant-related mortality (TRM) in 182 unrelated SCTs performed at a single center. We found association of polymorphism in the tumor necrosis factor alpha (TNF alpha) and interleukin-10 (IL-10) gene and TRM. Both the TNFd4 allele and the TNF alpha -1031C alleles are associated with high risk for TRM. Statistical analysis showed that both polymorphisms were present on a single haplotype. This haplotype was associated with high risk of TRM when present in recipient or donor, 55% (43%-67%) compared with 21% (12%-30%) when absent from both (P <.01). A further allele associated with this haplotype, TNFa5, is also associated with increased risk of TRM. For IL-10, presence of the donor R2-G-C-C haplotype was associated with decreased risk of TRM, 61% (43%-79%) versus 34% (25%-43%), P =.01. In contrast, possession of the R3-G-C-C haplotype by the donor predicted reduced risk of TRM, 30% (19%-41%, 95% CI) versus 53% (40%-66%, 95% CI), P =.01. No independent associations of cytokine polymorphisms with acute graft-versus-host disease were shown.

Rejection and recipient age.

In transplantation the risk of acute rejection decreases with recipient age. This is clearly illustrated in transplantation of a non-vascularised tissue, such as the cornea. In vascularised transplants, such as kidneys, acute rejection decreases with recipient age, but the phenomenon is obscured by the fact that chronic allograft nephropathy increases with age, and is further confounded by increased death from infectious disease and drug-related causes. The underlying cellular mechanisms responsible for this weakening of rejection are discussed, as is defective signal transduction leading to decreased activation of cells and decreased resistance to immunosuppressive drugs. This supports a view that less intensive immunosuppressive drug therapy is desirable in elderly recipients. Although pharmacokinetic studies are documented, there are no routine assays to measure efficacy of these drugs in individual patients. In summary, the decline in acute rejection with increasing recipient age may be due both to immunosenescence and decreased resistance to immunosuppressive drugs. Future assays to test these mechanisms could be used to tailor therapy to individual needs.

Quantitation of natural killer cell precursors in man.

A technique was developed to measure the frequency of natural killer cell precursors (NKpf) in human peripheral blood mononuclear cell (PBMC) samples. Functional maturity of NK cells was reflected in their ability to lyse target cells from the K562 cell line. During the development of the technique, venous blood was taken from one healthy adult and assayed at different times to avoid individual variation. The technique was based on the principle of limiting dilution analysis. The NKpf assay was set up with a range of cell dilutions from 40,000 to 625 per 100 microl/well in 96-well culture plates. At the end of the culture period, the K562 cell line labelled with europium (Eu-K562) was added and the Eu-release was measured in culture supernatants using time-resolved fluorometry. The NKpf value differed between individuals and was influenced by the length of time in culture, being maximal at day 5. Maturation of NKp required the continuous presence of recombinant interleukin 2 (rIL-2), or rIL-15, both being equally effective. In the absence of cytokines, the functional NK cells declined rapidly beyond 24 h in culture. Irradiated allogeneic cells appeared to substitute in part for cytokines, but the numbers of allo-activated NKpf were lower than those observed when allo-activated NKpf were cultured with rIL-2. This suggested selective activation by the allogeneic stimulus of subsets of NKp or rIL-2-rescue of NKp subsets destined for apoptotic cell death. Alternatively, the increased frequency could have been attributable to activation of precursors of natural killer-T cells (NK-Tp). This assay is suitable for estimating the total number of precursors of functional NK cells in the blood of patients.