Targeting complement at the time of transplantation.

Complement activation occurs in at least two phases when an organ is transplanted into a naive recipient: during reperfusion with recipient blood particularly when the donor organ has undergone a significant period of ischaemia and then during acute rejection once the recipient immune system has recognised the donor tissue as non-self. Both of these reactions are most obvious in the extravascular compartment of the transplanted organ and involve local synthesis of some of the key complement components as well as loss of controls that limit the activation of the pivotal component C3. In contrast, sensitised individuals with pre-existing circulating antibodies have an immediate reaction against the transplant organ that is also complement dependent but is enacted in the intravascular space. All three types of injury (ischaemia-reperfusion, acute rejection, hyperacute rejection) have a critical effect on transplant outcome. Here we discuss therapeutic strategies that are designed to overcome the impact of these factors at the start of transplantation with the aim of improving long-term transplant outcomes. These include the concept of treating the donor organ with modified therapeutic regulators that are engineered to be retained by the donor organ after transplantation and prevent inflammatory injury during the critical early period. By targeting the donor organ with anchored therapeutic proteins, the systemic functions of complement including host defence remain intact. The control of complement activation during the first stages of transplantation, including the possibility that this will reduce the capacity of the graft for stimulating the adaptive immune system, offers an important prospect for increasing the longevity of the transplant and offsetting demand on the limited supply of donor organs. It also provides a model in which the benefits and indications for localised therapy to maximise therapeutic efficiency and minimise the systemic disturbance may be instructive in other complement-related disorders.

Comparison of mycophenolic acid concentrations determined by a new PETINIA assay on the dimension EXL analyzer and a HPLC-UV method.

OBJECTIVE: Mycophenolic acid requires routine therapeutic drug monitoring. We evaluated the suitability of a new PETINIA (particle enhanced turbidimetric inhibition immunoassay) assay on the Dimension EXL analyzer for monitoring of mycophenolic acid by comparing values obtained by this assay with a HPLC-UV method. DESIGN AND METHODS: Mycophenolic acid concentrations determined in sera of 60 organ transplant recipients using high performance liquid chromatography combined with ultraviolet detection (HPLC-UV, reference method) and the new immunoassay on the Dimension RxL analyzer. RESULTS: The within and between run precision of the new PETINIA assay was <10%. The assay was linear for a mycophenolic acid concentration up to 30µg/mL. When mycophenolic acid concentrations in 60 transplant recipients obtained by the HPLC-UV (x-axis) method were compared with corresponding values obtained by the PETINIA assay (y-axis), the following regression equation was obtained: y=1.1204 x+0.0881 (r=0.983, n=60). CONCLUSIONS: If PETINIA assay is used for therapeutic drug monitoring of mycophenolic acid, caution must be exercised in interpreting serum mycophenolic acid level due to observed positive bias.

Immunologic challenges in small bowel transplantation.

Since the introduction of tacrolimus, small-bowel and multivisceral transplantion has increased to 100-200/year in the United States. The intestine carries more passenger lymphocytes than other organs, and bidirectional trafficking of lymphocytes and other immunocytes begins as soon as the vascular clamp is released. Because of ischemia-reperfusion injury and exposure to ligands for Toll-like receptors from the lumen, the innate immune system of the graft is activated, causing inflammation which must be brought under control by regulatory cells. Inclusion of the liver in the allograft favors graft acceptance, but the mechanism of this effect has not been determined. Anti-HLA and other anti-donor antibodies clearly play a major role in determining the long-term fate of the graft, as reflected in 5-year graft survival. Development of new (de novo) HLA antibodies and/or increases in their titers or function-especially the ability to bind C1q and activate complement increase the risk of graft loss. Monitoring antidonor antibody production and the use of new therapies including complement inhibitors will contribute to increasing success of SBT.

Natural killer cell immunity after transplantation.

Transplantation immunology has traditionally focused on adaptive, i.e., T- and B-cell reactions. More recently, natural killer (NK) cells were also recognised as playing an important role after transplantation of solid organs and haematopoietic stem cells. NK cells recognise "cell stress" induced by viral replication and tumour transformation via activating receptors, and are negatively regulated by the interaction between inhibitory molecules and autologous human leukocyte antigens (HLA). The most important inhibitory molecules belong to the family of killer cell immunoglobulin-like receptors (KIR). Differences in the inhibitory KIR/HLA interaction between stem cell donor and patient may lead to beneficial NK cell alloreactivity, resulting in specific graft-versus-tumour reactions, which occur in the absence of graft-versus-host disease. The immaturity of NK cells produced by the stem cell graft early after transplantation has led to different approaches of adoptive transfer of NK cells to further increase tumour control. The function and role of activating KIR receptors is less clear. Recent data have suggested, that activating KIR may also contribute to anti-tumour immunity after stem cell transplantation, as patients transplanted from donors carrying high numbers of activating KIR receptor genes show reduced relapse rates. In particular, protection from post-transplant disease relapse was demonstrated in transplants carried out from donors carrying the activating KIR2DS1 receptor, if the recipients also expressed the KIR2DS1 ligand HLA-C2. In conclusion, NK cells have been firmly established in the last two decades as relevant players in transplant immunology, which can critically determine the outcome of haematopoietic stem cell grafts.

The pathophysiology of endothelin in complications after solid organ transplantation: a potential novel therapeutic role for endothelin receptor antagonists.

Although short-term allograft survival after solid organ transplantation has improved during the past two decades, improvement in long-term graft survival has been less pronounced. Common complications after transplantation include chronic allograft rejection, nephrotoxicity from calcineurin inhibitors (CNIs), and systemic hypertension, which all impact posttransplantation morbidity and mortality. Endothelin (ET)-1, a potent endogenous vasoconstrictor, inducer of fibrosis, and vascular smooth muscle cell proliferation, may play a key role in both the development of CNI-induced nephrotoxicity and endothelial vasculopathy in chronic allograft rejection. ET-1 levels increase after isograft implantation, and ET-1 plays a key role in CNI-induced renal vasoconstriction, sodium retention, and hypertension. Preclinical studies have demonstrated that endothelin receptor antagonists (ERAs) can reduce or prevent CNI-induced hypertension after renal transplantation. In addition, ERAs can ameliorate CNI-induced renal vasoconstriction and improve proteinuria and preserve renal function in animal models of renal transplantation. ET-1 may also play a significant role in cardiac allograft vasculopathy, and in animal models, ERAs improve pulmonary function and ischemic-reperfusion injury in lung transplantation and hepatic function and structure in liver transplantation. Emerging pharmacokinetic data suggest that the selective ERA ambrisentan may be used safely in conjunction with the most commonly used immunosuppressive agents tacrolimus and mycophenolate, albeit with appropriate dose adjustment. The weight of available evidence pointing toward a potential beneficial role of ERAs in ameliorating common complications after solid organ transplantation must be balanced with potential toxicities of ERAs but suggests that a randomized clinical trial of ERAs in transplant patients is warranted.

Intestinal regeneration, residual function and immunological priming following rescue therapy after rat small bowel transplantation.

Clinical evidence suggests that recurrent acute cellular rejection (ACR) may trigger chronic rejection and impair outcome after intestinal transplantation. To test this hypothesis and clarify underlying molecular mechanisms, orthotopic/allogenic intestinal transplantation was performed in rats. ACR was allowed to occur in a MHC-disparate combination (BN-LEW) and two rescue strategies (FK506monotherapy vs. FK506+infliximab) were tested against continuous immunosuppression without ACR, with observation for 7/14 and 21 days after transplantation. Both, FK506 and FK506+infliximab rescue therapy reversed ACR and resulted in improved histology and less cellular infiltration. Proinflammatory cytokines and chemotactic mediators in the muscle layer were significantly reduced in FK506 treated groups. Increased levels of CD4, FOXP3 and IL-17 (mRNA) were observed with infliximab. Contractile function improved significantly after FK506 rescue therapy, with a slight benefit from additional infliximab, but did not reach nontransplanted controls. Fibrosis onset was detected in both rescue groups by Sirius-Red staining with concomitant increase of the fibrogenic mediator VEGF. Recovery from ACR could be attained by both rescue therapy regimens, progressing steadily after initiation of immunosuppression. Reversal of ACR, however, resulted in early stage graft fibrosis. Additional infliximab treatment may enhance physiological recovery of the muscle layer and enteric nervous system independent of inflammatory reactions.

The use of micro pulse oximetery as a new detector of tissue perfusion in solid organ transplantation.

Vascular complications are a frequent cause of transplant failure; angiography, duplex sonography, computerized tomography (CT) scan, CT-angiography and microdialysis are the methods that were suggested for the detection of arterial obstruction after transplantation. In this study, we suggest a new method. Eight healthy adult dogs were included in the trial. All cases were operated by the same surgeon and the liver, pancreas, spleen, kidney and bowel tissue were exposed. The probes of the device, which were designed for this study, were inserted on the organ parenchyma. The device, a neonatal pulse oximeter, has two probes that were fixed by a holder in front of each other; the distance between the probes was changeable via a spring. The pulse and the oxygen saturation of the tissue were measured initially. Following this, by inducing ischemia with vessel clamping, the pulse and the oxygen saturation were measured again. The collected data were analyzed under the supervision of a statistician. In the liver and spleen, we could not detect a clear pulse wave and oxygenation. On the other hand, in the pancreas, kidney and bowel, we detected a clear curve of oxygenation and pulse in all cases. Obstruction caused significant changes: the pulse was not detected and the oxygenation decreased significantly. Our study suggests that with early diagnosis, the surgeons can detect arterial occlusion immediately and early intervention may decrease parenchymal damage. This study is the first experience in this field, and these findings need to be validated with further studies.

Hypertension in solid organ transplant recipients.

Hypertension (HT) is one of the most frequent complications of solid organ transplantation; about 70-90% of this population have high blood pressure or require antihypertensive therapy. Abnormal blood pressure is a potent non-immunological risk factor directly related to patient and graft survival. The etiology of hypertension after orthotopic heart transplantation is multifactorial and varies depending on the time following transplantation. In the early period after transplantation, hypertension is generally related to intravascular volume expansion and persistently increased systemic vascular resistance. Other factors predominant in kidney allograft recipients include: donor age, donor familial history of hypertension, transplant renal artery stenosis, graft function, the recurrence or de novo appearance of glomerulonephritis in transplanted kidney, and post-biopsy arteriovenous fistula. In liver and heart transplantation, hypertension is mainly due to impaired kidney function, with all its consequences. Another contributing factor is immunosuppressive regimen based on calcineurin inhibitors and steroids. The management of post-transplant hypertension usually requests non-pharmacological and pharmacological treatment. In this review, the pathogenesis and treatment of post-transplant hypertension in solid organ transplantation is presented.

Organ transplantation in the 21th century.

Mankind has always been interested in investigating and searching for solutions regarding the body deterioration due to factors such as disease, damage caused by trauma, toxins or radiation or just the process of ageing. Here, we summarize the history of scientific advances in solid organ transplantation, in the areas strictly linked to transplantation. The period between the end of the 19th and beginning of the 20th century has been called by some authors the "Era of allografting". This was a muddled period with many studies and publications on very diverse transplants, from Kocher's (Nobel Prize in 1909) who transplanted thyroid extracts, to Brown-Sequard who tried to rejuvenate people by using grafts of guinea pig testicle extracts. In the midst of the 20th century, Sir Medawar pointed out that the rejection of transplant organs by the recipient body was mediated by an immunological reaction, which should be modified. Since then, there has been an open period of discovery of new immunosuppressive drugs which have revolutionised the outcomes of solid organ transplantations. New challenges have appeared over the last few years, these efforts have focused on the search to extend graft durability and with it recipient patient survival times, as well as improve their quality of life.

Immunology and the challenge of transplantation.

Transplantation of tissues or organs between individuals who are not genetically related often leads to rejection by the recipient. The human genes responsible for this process are located on the short arm of the chromosome 6 and are called Major Histocompatibility Complex (MHC). Six main loci have been identified in the human MHC: HLA-A, HLA-B and HLA-C belong to the HLA class I, while HLA-DP, HLA-DQ and HLA-DR belong to HLA class II. The physiological function of MHC molecules is to present peptides to the T cells. Indeed, they are integral components of the ligands that recognise most T cells, since the receptor of the T cell (TCR) has specificity for complexes of foreign antigenic peptides, and self-MHC molecules. Thus the proteins of the MHC are responsible for the body being able to distinguish between its own and foreign cells, known as self-tolerance and consequently are the proteins which determine the evolution of transplants. The special case of foreign MHC antigen recognition is known as allorecognition and consists of the capacity of T cells to recognise peptide/MHC complexes with which they have not been in contact during the process of maturation in the thymus. There are two mechanisms of allorecognition, direct and indirect; both can lead to rejection of the transplant. Direct recognition prevails during the first few weeks or months after transplantation, and is caused by the APCs of the donor. These cells start disappearing from the transplanted organ and indirect recognition becomes important. There is evidence that the indirect pathway is sufficient to mediate both acute and chronic rejection. In this chapter we will describe fundamental aspects of the MHC system, as well as, specifically, its involvement in the allogenic response of the immune system against organ transplants.

Comparative study on signal transduction in endothelial cells after anti-a/b and human leukocyte antigen antibody reaction: implication of accommodation.

BACKGROUND: Recent development of immunosuppressive therapy has provided a platform for clinical human leukocyte antigen (HLA)- and ABO-incompatible kidney transplantation. However, the prognosis seems to be different between the two. Accommodation, the condition of no injury even in the presence of antidonor antibody, is one of the key factors for successful transplantation with antidonor antibody. The purpose of this study was to compare signal transduction between anti-A/B and anti-HLA antibody reaction and to elucidate the mechanisms underlying accommodation. METHODS: Blood type A- or B-transferase gene was transfected into human EA.hy926 endothelial cells. After cell sorting, A- or B-expressing cells at high levels were obtained. The effects of anti-HLA and anti-A/B antibody binding on complement-mediated cytotoxicity and signal transduction were examined. RESULTS: Preincubation with anti-HLA antibodies only at low levels (<10% of saturation level) or anti-A/B antibodies at high levels (even at near saturation levels) for 24 hr resulted in resistance to complement-mediated cytotoxicity. Anti-A/B antibody ligation inactivated ERK1/2 pathway and increased complement regulatory proteins such as CD55 and CD59, whereas anti-HLA ligation activated PI3K/AKT pathway and increased cytoprotective genes such as hemeoxygenase-1 and ferritin H. CONCLUSION: Complement inhibition by upregulation of CD55 and CD59 through ERK1/2 inactivation might play a substantial role in accommodation after ABO-incompatible transplantation, which could also explain the intriguing finding of C4d deposition in the graft without rejection.

Use of RNA interference to minimize ischemia reperfusion injury.

RNA interference (RNAi) is an endogenous mechanism of cellular RNA control through degradation of specific messenger RNA sequences. This process of gene silencing may be exploited by the use of small interfering RNA (siRNA) to mediate precise control of targeted cellular functions. The nature of transplantation leads invariably to tissue injury, as organs are damaged by the loss of blood supply and resultant ischemia associated with the procurement procedure. Upon reperfusion, an inflammatory program is activated, and subsequent injury results in delayed graft function and, potentially, organ failure. Many of the molecular components in ischemia-reperfusion injury (IRI) have been identified, but effective therapeutics are not currently available. Accumulating evidence supports a role for siRNA in controlling IRI, as siRNA is specific, relatively low in toxicity, and limited in duration of effect. The capacity of siRNA to control IRI-related transcription factors, cell death and apoptosis, complement factors, and oxidative stress molecules supports the concept that RNAi-based therapeutics represent a novel and promising strategy for the control of IRI. However, there are issues of RNAi strategies, including siRNA design, "off-target" effects, and delivery that merit consideration in approaching IRI with gene silencing. This review will provide an overview of current concepts in RNAi and the potential application to IRI in solid organ transplantation.

Mechanisms of rejection: current perspectives.

Rejection is the major barrier to successful transplantation. The immune response to an allograft is an ongoing dialogue between the innate and adaptive immune system that if left unchecked will lead to the rejection of transplanted cells, tissues, or organs. Activation of elements of the innate immune system, triggered as a consequence of tissue injury sustained during cell isolation or organ retrieval and ischemia reperfusion, will initiate and amplify the adaptive response. T cells require a minimum of two signals for activation, antigen recognition, and costimulation. The activation requirements of naive T cells are more stringent than those of memory T cells. Memory T cells are present in the majority of transplant recipients as a result of heterologous immunity. The majority of B cells require help from T cells to initiate antibody production. Antibodies reactive to donor human leukocyte antigen molecules, minor histocompatibility antigens, endothelial cells, RBCs, or autoantigens can trigger or contribute to rejection early and late after transplantation. Antibody-mediated rejection triggered by alloantibody binding and complement activation is recognized increasingly as a significant contribution to graft loss. Even though one component of the immune system may dominate and lead to rejection being described in short hand as T cell or antibody mediated, it is usually multifactorial resulting from the integration of multiple mechanisms. Identifying the molecular pathways that trigger tissue injury, signal transduction and rejection facilitates the identification of targets for the development of immunosuppressive drugs.

Is the knowledge on tissue microcirculation important for microsurgeon?

This review article outlines the importance of knowledge on the hemodynamics of microcirculatory responses during free tissue transfer procedures. Anatomy and pathophysiology of peripheral microcirculation are outlined in the context of its responses during microsurgical procedures submitted to ischemia and reperfusion injury. The factors influencing the patient's outcome such as neural, humoral, and muscular regulations and prostoglandins, kinins, nitric oxide actions, and so on are outlined. In addition, other important factors influencing microcirculatory responses are discussed. The goal of this review article is to introduce nonsurgical factors independent of the microsurgeon's control which, via changes in microcirculatory hemodynamics, may contribute to free flap survival and final patient's outcomes. Thus, we hope that this overview of the pathophysiology of tissue microcirculation will help microsurgeons to monitor factors beyond control of vessel patency and technical aspects of microvascular anastomosis.

Organ donation in Switzerland: a survey on marginal or extended criteria donors (ECD) from 1998 to 2009.

BACKGROUND: The widening gap between the numbers of patients on the waiting list for organ transplantation and the insufficient numbers of organ donors results in the use of "critical" donors, so-called marginal donors or extended criteria donors. Data concerning the evaluation of extended criteria donors (ECD) in Switzerland are sparse. METHODS: All organ donors in Switzerland between 1.1.1998 and 30.6.2009 have been evaluated for special criteria. ECD were defined on the basis of at least one of seven criteria: six DOPKI criteria (ECD-DOPKI) and/or age ≥60 yr (ECD-Age). Once included in the study, special features, short time follow-up (first 7 days after transplantation) and the cold ischaemia time of all the transplanted organs were evaluated. RESULTS: During the period 1.1.1998 to 30.6.2009, a total of 408 organ donors were classified as ECD, reflecting 39% of all organ donors in this time period. Despite the fact that all organ donors in this study fulfilled at least one inclusion criterion, the number of recipients with satisfactory primary organ function was always higher than the respective number with a negative primary outcome within the first seven days after transplantation. A longer cold ischaemia time was associated with organs showing insufficient primary organ function compared to organs with satisfactory primary function. A relevant causal relationship cannot be investigated on the basis of our limited data. In addition, a longer observation period would be necessary to draw a more precise conclusion. CONCLUSIONS: ECD as defined by DOPKI and/or age represent a high proportion of all organ donors in Switzerland but show a remarkably good outcome.

Long-term nutrition and predictors of growth and weight gain following pediatric intestinal transplantation.

BACKGROUND: Advances in intestinal transplantation (ITx) have resulted in improved survival and the opportunity to examine nutritional outcomes. The aim of this study was to describe detailed, long-term nutritional results and identify positive predictors of growth and weight gain following pediatric ITx. METHODS: A single-center retrospective, Institutional Review Board-approved review of a prospective database was conducted. Inclusion criteria were ITx recipients 18 years or younger with survival of 6 months or more. Outcomes included anthropometric measurements and biochemical markers at 6, 12, 24, 36, and 48 months post-ITx. More than 25 ITx-related variables were analyzed as potential predictors of growth and weight gain. Statistical analysis was performed using chi-square test, t test, and analysis of variance. RESULTS: Between November 1991 and April 2007, 50 children received 55 ITx; 33 patients met eligibility criteria. Median age at ITx was 2.2 years, follow-up time was 3.8 years, and time from ITx to cessation of total parenteral nutrition was 31 days. The most common micronutrient deficiencies post-ITx were zinc, iron, and copper. Serum protein levels improved significantly over time. Weight gain occurred within 6 months and vertical growth within 12 months, although limited catch-up growth was seen. Early predictors of weight gain and growth included shorter hospitalization and absence of rejection. Long-term predictors were low steroid dosage, infrequent hospitalization, and the use of peptide-based formulas. CONCLUSIONS: This represents one of the largest and most comprehensive long-term studies on nutritional outcomes in pediatric ITx. Overall, positive growth and weight gain were seen as were micronutrient deficiencies. Numerous long-term nutritional challenges exist which require a multidisciplinary approach and future prospective studies.