Dissertations / Theses on the topic 'Immunosuppression'

This research is part of an effort to develop non-mammalian surrogate immunoessays with the earth worm Lumbricus terrestris to assess immunotoxic potential of xenobiotics to mammals. The objective was to determine if earthworm immunoessays, namely E- and S- rosette formation and phagocytosis, are sensitive to a known mammalian immunotoxin, the PCB Arclor 1254. Results are presented in terms of PCB exposure and tissue concentrations during uptake/depuration.

Les travaux de l'équipe ont permis de montrer qu'IL4I1 est une L-amino acide oxydase sécrétée par les cellules d'origine myéloïde dégradant la phénylalanine en H2O2, NH3 et phénylpyruvate. Elle est fortement exprimée au sein des tumeurs humaines et facilite l'échappement tumoral dans un modèle de mélanome murin. Cette enzyme inhibe l'expression de la chaîne ζ du TCR ainsi que la prolifération des lymphocytes T effecteurs/mémoires via la production d'H2O2. IL4I1 appartient donc à une famille d'enzymes régulatrices des réponses immunitaires impliquées dans la défaillance de la réponse anti-tumorale.Au cours de ma thèse, j'ai montré qu'IL4I1 induit la différenciation des lymphocytes T CD4+ naïfs conventionnels en cellules CD25fortFoxP3+ chez l'Homme et la souris. Ces cellules exercent une action suppressive in vitro équivalente à celle de cellules régulatrices obtenues sans IL4I1 et leur phénotype est similaire. La promotion de la différenciation Treg par IL4I1 a pu être observée dans différentes conditions in vitro et s'avère particulièrement importante lorsque les cellules sont cultivées sans ajout d'IL2 et de TGFβ. Le mécanisme impliqué reposerait en partie sur la consommation de Phe par l'activité enzymatique qui serait responsable de l'inhibition de la voie mTORC1 observée.En conclusion, nous avons démontré un nouveau rôle d'IL4I1 sur les lymphocytes T. Ainsi, en inhibant la prolifération des lymphocytes T et en induisant la polarisation Treg, IL4I1 pourrait jouer un rôle important dans l'échappement tumoral. IL4I1 étant sécrétée et peu exprimée à l'état physiologique, elle pourrait être la cible de traitements adjuvants dans le cancer
Our team has shown that IL4I1 is a secreted L-amino acid oxidase which degrades phenylalanine into H2O2, NH3 and phenylpyruvate.. This enzyme is produced by myeloid cells and expressed within human cancers. IL4I1 expression facilitates tumor growth in a mouse model. IL4I1 inhibits TCRζ chain expression and T lymphocyte proliferation via H2O2 production. Therefore IL4I1 belongs to a family of enzymes endowed with immune regulatory functions involved in the anti-tumor response failure.During my PhD, I showed that IL4I1 induces CD25highFoxP3+ cells differentiation from conventional naïve CD4+ T cells, both in humans and mice in vitro systems. These cells exert similar in vitro suppressive activity than those obtained without IL4I1 with a similar phenotype. Treg differentiation promotion by L4I1 is observed in various in vitro conditions and is particularly important when cells are cultured without addition of IL2 and TGFβ. The involved mechanism would partially depend on the phenylalanine consumption by the enzymatic activity which would be responsible for the mTORC1 pathway inhibition observed.In conclusion, we have demonstrated a new mechanism of IL4I1 action on T lymphocytes. Thus, by inhibiting T lymphocytes proliferation and by inducing Treg polarization, IL4I1 could play an important role in tumor escape. Since IL4I1 is secreted and weakly expressed under physiological conditions, it could be the target of adjuvant therapy in cancer

Corneal transplantation is performed to restore vision or to relieve pain in patients with damaged or diseased corneas. However, approximately 40% of corneal allografts fail after 10 years. The most common cause of graft failure is irreversible immunological rejection, primarily mediated by CD4+ T cells, despite the topical application of glucocorticosteroids. The aim of this project was to investigate the anatomic site of antigen presentation during corneal transplantation in the rat, by using a lentiviral vector to express an anti-CD4 antibody fragment at potential sites of antigen presentation, including the donor corneal endothelium, the anterior segment of the eye and the cervical lymph nodes. Dual-gene lentiviral vectors were constructed by inserting the 2A self-processing sequence between two transgenes. This allowed expression of two transgenes within a single open reading frame. In vitro characterisation of the dual-gene vectors was performed in cell culture experiments, which showed that transgenic proteins were expressed at lower levels from dual-gene vectors compared to the expression from single-gene vectors and expression was lowest when the transgene was situated downstream of the 2A self-processing sequence. To locate the anatomic site of antigen presentation during corneal transplantation in rats, a lentiviral vector carrying an anti-CD4 antibody fragment was delivered to the corneal endothelium either immediately prior to corneal transplantation by ex vivo transduction of the donor corneas, or 5 days prior to corneal transplantation by anterior chamber injection into both the recipient and the donor rats. A separate group of recipient rats received intranodal injections of the lentiviral vector carrying an anti-CD4 antibody fragment into the cervical lymph nodes 2 days prior to corneal transplantation. Another group of rats underwent bilateral lymphadenectomy of the cervical lymph nodes 7 days prior to corneal transplantation. Corneal allografts were scored daily for opacity, inflammation and neovascularisation. Expression of the anti-CD4 antibody fragment from transduced tissues was detected using flow cytometry and polymerase chain reaction. Modest, but significant prolongation of corneal allograft survival was experienced by rats that received ex vivo transduction of the donor corneas with a lentiviral vector carrying an anti-CD4 antibody fragment immediately prior to corneal transplantation, but all grafts did eventually reject. Anterior chamber injection of the lentiviral vector carrying the anti-CD4 antibody fragment 5 days prior to corneal transplantation into both recipient and donor eyes did not prolong allograft survival. Intranodal injection of a lentiviral vector carrying an anti-CD4 antibody fragment did not prolong the survival of the corneal allografts, nor did bilateral lymphadenectomy of the cervical lymph nodes 7 days prior to corneal transplantation. Neither expression of the anti-CD4 antibody fragment in the cervical lymph nodes nor the removal of these nodes was able to prolong corneal allograft survival in rats, suggesting that T cell sensitisation could potentially occur elsewhere in the body. However, expression of the anti-CD4 antibody fragment from the donor corneal endothelium was able to prolong corneal allograft survival, suggesting that some antigen presentation might occur within the anterior segment of the eye. Based on the findings described in this thesis and those of others, I propose that antigen presentation in the rat occurs within anterior segment of the eye and within the secondary lymphoid tissues such as the cervical lymph nodes, and that inhibiting antigen presentation at one of these sites will delay graft rejection. However, to completely abolish antigen presentation during corneal transplantation in the rat, I hypothesise that antigen presentation within both the anterior segment of the eye and within the secondary lymphoid tissues must be inhibited.

Les cellules souches mésenchymateuses (CSM) sont des cellules stromales présentes dans différents types de tissus. En plus de leur capacité à se différencier en plusieurs lignées (chondrocytes, adipocytes et ostéoblastes), les CSM présentent également des propriétés immunosuppressives. Bien que ces mécanismes soient loin d'être entièrement compris, leur capacité immunosuppressive a récemment été démontrée comme étant modulée par des miARN. L'arthrose est la forme la plus courante de maladies articulaires sans traitement curatif et se caractérise principalement par la dégradation du cartilage articulaire, avec des altérations osseuses sous-chondrales et une inflammation synoviale. Les CSM pourraient offrir un potentiel thérapeutique intéressant pour le traitement de l'arthrose.Nos travaux ont montré qu'une injection autologue de CSM d'origine adipeuse (ASC) dans une articulation arthrosique améliore la douleur et les niveaux fonctionnels chez les patients. Nous avons souligné la tolérance immunitaire systémique induite à la suite d'injections intra-articulaires d'ASC. Enfin, nous avons étudié le profil d'expression des miARN des CSM humaines lors de leur stimulation par des cellules mononuclées du sang préalablement activés. Nous avons identifié le miR-29a et le PSAT1 comme de nouveaux candidats pour réguler l'activité immunosuppressive médiée par les CSM
Mesenchymal Stem Cells (MSCs) are stromal cells present in a number of different tissue types. In addition to their ability to differentiate into multiple lineages (chondrocytes, adipocytes and osteoblasts), MSCs also display immunosuppressive properties. Whilst these mechanisms are far from fully understood, their immunosuppressive capacity has recently been shown to be modulated by miRNAs. OA is the most common form of joint diseases without curative treatment and mainly characterized by the degradation of articular cartilage, with subchondral bone alterations and synovial inflammation. MSC might provide therapeutic potential for treatment of OA.Here, we showed that an autologous injection of adipose-derived MSC (ASC) into an osteoarthritic joint improved pain and function levels in patients. We underscored the systemic immune tolerance induced following intra-articular injections of ASCs. Finally, we investigated the miRNA expression profile of human MSCs upon their stimulation by peripheral blood mononuclear cells. We identified miR-29a and PSAT1 as new candidates to regulate immunosuppressive activity mediated by MSCs

Thesis (Ph. D.)--University of Sydney, 2006.
Title from title screen (viewed 21 May 2007). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Public Health, Faculty of Medicine. Includes bibliographical references. Also available in print form.

The societal burden of autoimmune diseases and donor organ transplant rejection in developed countries reflects the lack of effective immune suppressive drugs. The main objective of my thesis was to develop novel fusion proteins targeting receptors linked to autoimmunity; strategies that will allow the suppression of autoreactive cells while sparing resting lymphocytes. Interleukin (IL) 15 has been demonstrated to exert its effects mainly on activated T-cells triggered via their T-cell receptor (TCR). Since we found that the fusion of granulocyte-macrophage colony stimulating factor (GMCSF) to IL15 - aka GIFT15 - paradoxically leads to aberrant signalling downstream of the IL15R and blocks interferon (IFN)-gamma secretion in a mixed lymphocyte reaction (MLR), we hypothesized to use this fusokine in proof-of-principle cell transplantation models and shown that GIFT15 can indeed block the rejection of allogeneic and xenogeneic cells in immunocompetent mice. Additionally, we found that ex vivo GIFT15 treatment of mouse splenocytes lead to the generation of regulatory B-cells (Bregs). These Bregs express high levels of MHCII, IL10 and are capable to block antigen (Ag)-presentation in vitro as third party bystander cells. Moreover, a single injection of these GIFT15-generated Bregs in mice with pre-developed experimental autoimmune encephalomyelitis (EAE) leads to long lasting remission of disease.
Along those lines, we also found that mesenchymal stromal cells (MSCs) lead to the paracrine conversion of CCL2 to an antagonist form capable of specifically inhibiting plasma cells and activated Th17 cells. This mechanistic insight informed the design of a second class of suppression fusokine. Namely, the fusing of antagonist CCL2 to GMCSF - aka GMME1. We tested its potential use in autoimmune diseases such as EAE and rheumatoid arthritis (RA). We demonstrated that GMME1 leads to asymmetrical signalling and inhibition of plasma cells as well as Th17 EAE/RA-reactive CD4 T-cells. The net outcome of these pharmacological effects is the selective depletion of CCR2-reactive T-cells as demonstrated both in vitro and in vivo.
Overall, our data support the use of our fusion proteins as part of a powerful and specific immunosuppressive strategy either as directly injectable protein biopharmaceuticals or through the ex vivo generation of autologous Bregs in the case of GIFT15.

Introduction Kidney transplantation confers both survival and quality of life advantages over dialysis for most people with end-stage kidney disease (ESKD). The mortality rate on dialysis is 10-15% per year, compared with 2-4% per year post-transplantation. Short-term graft survival is related to control of the acute rejection process, requiring on-going immunosuppression. Most current immunosuppressive algorithms include one of the calcineurin inhibitors (CNI: cyclosporin or tacrolimus), an anti-metabolite (azathioprine or mycophenolate) and corticosteroids, with or without antibody induction agents (Ab) given briefly peri-transplantation. Despite this approach, between 15-35% of recipients undergo treatment for an episode of acute rejection (AR) within one year of transplantation. Transplantation is not without risk, and relative mortality rates for kidney recipients after the first post-transplant year remain 4-6 times that of the general population. Longer-term transplant and recipient survival are related to control of chronic allograft nephropathy (rooted in the interplay of AR, non-immunological factors, and the chronic nephrotoxicity of CNI) and limitation of the complications of chronic ESKD and long-term immunosuppression: cardiovascular disease, cancer and infection, which are responsible for 22%, 39% and 21% of deaths respectively. This thesis is presented as published works on the theme of immunosuppression and cancer after kidney transplantation. The work presented in the first chapters of this thesis has striven to identify, evaluate, synthesise and distil the entirety of evidence available of new and established immunosuppressive drug agents through systematic review of randomised trial data, with particular emphasis on quantifying harms of treatment. The final chapters use inception cohort data from the Australian and New Zealand Dialysis and Transplant Registry (ANZDATA), which is first validated then used to explore the risk of cancer in more detail than was possible from trial data alone. Interleukin 2 receptor antagonists Interleukin-2 receptor antagonists (IL2Ra, commercially available as basiliximab and daclizumab) are humanised or chimeric IgG monoclonal antibodies to the alpha subunit of the IL2 receptor present only on activated T lymphocytes, and the rationale for their use has been as induction agents peri-transplantation. Introduced in the mid-1990s, IL2Ra use has increased globally, and by 2003 38% of new kidney transplant recipients in the United States and 25% in Australasia received an IL2Ra. This study aimed to systematically identify and synthesise the evidence of effects of IL2Ra as an addition to standard therapy, or as an alternative to other induction agents. We identified 117 reports from 38 randomised trials involving 4893 participants. Where IL2Ra were compared with placebo (17 trials; 2786 patients), graft loss was not different at one (Relative Risk -RR 0.84; 0.64 to 1.10) or 3 years (RR 1.08; 0.71 to1.64). AR was reduced at 6 months (RR 0.66; 0.59 to 0.74) and at 1 year (RR 0.66; 0.59 to 0.74) but cytomegalovirus (CMV) disease (RR 0.82; CI 0.65 to 1.03) and malignancy (RR 0.67; 0.33 to1.36) were not different. Where IL2Ra were compared with other antibody therapy no significant differences in treatment effects were demonstrated, but IL2Ra had significantly fewer side effects. Given a 40% risk of rejection, 7 patients would need treatment with IL2Ra in addition to standard therapy, to prevent 1 patient having rejection, with no definite improvement in graft or patient survival. There was no apparent difference between basiliximab and daclizumab. Tacrolimus versus cyclosporin for primary immunosuppression There are pronounced global differences in CNI use; 63% of new kidney transplant recipients in the USA but only 22% in Australia receive tacrolimus as part of the initial immunosuppressive regimen. The side effects of CNI differ: tacrolimus is associated more with diabetes and neurotoxicity, but less with hypertension and dyslipidaemia than cyclosporin, with uncertainty about equivalence of nephrotoxicity or how these relate to patient and graft survival, or impact on patient compliance and quality of life. This study aimed to systematically review and synthesise the positive and negative effects of tacrolimus and cyclosporin as initial therapy for renal transplant recipients. We identified 123 reports from 30 randomised trials involving 4102 participants. At 6 months graft loss was reduced in tacrolimus-treated recipients (RR 0•56; 0•36 to 0•86), and this effect persisted for 3 years. The relative reduction in graft loss with tacrolimus diminished with higher levels of tacrolimus (P=0.04), but did not vary with cyclosporin formulation (P=0.97) or cyclosporin level (P=0.38). At 1 year, tacrolimus patients suffered less AR (RR 0•69; 0•60 to 0•79), and less steroid-resistant AR (RR 0•49; 0•37 to 0•64), but more insulin-requiring diabetes (RR 1•86; 1•11 to 3•09), tremor, headache, diarrhoea, dyspepsia and vomiting. The relative excess in diabetes increased with higher levels of tacrolimus (P=0.003). Cyclosporin-treated recipients experienced significantly more constipation and cosmetic side-effects. We demonstrated no differences in infection or malignancy. Treating 100 recipients with tacrolimus instead of cyclosporin for the 1st year post-transplantation avoids 12 suffering acute rejection and 2 losing their graft but causes an extra 5 to become insulin dependent diabetics, thus optimal drug choice may vary among patients. Target of rapamycin inhibitors for primary immunosuppression Target of rapamycin inhibitors (TOR-I) are among the newest immunosuppressive agents and have a novel mode of action but uncertain clinical role. Sirolimus is a macrocyclic lactone antibiotic and everolimus is a derivative of sirolimus. Both prevent DNA synthesis resulting in arrest of the cell cycle. Animal models suggested TOR-I would provide synergistic immunosuppression when combined with CNI, but early clinical studies demonstrated synergistic nephrotoxicity. Since then diverse trials have explored strategies that avoid this interaction and investigated other potential benefits. The aim of this study was to systematically identify and synthesise available evidence of sirolimus and everolimus when used in initial immunosuppressive regimens for kidney recipients. We identified 142 reports from 33 randomised trials involving 7114 participants, with TOR-I evaluated in four different primary immunosuppressive algorithms: as replacement for CNI, as replacement for antimetabolites, in combination with CNI at low and high dose, and with variable dose of CNI. When TOR-I replaced CNI (8 trials, 750 participants), there was no difference in AR (RR 1.03; 0.74 to 1.44), but creatinine was lower (WMD -18.31 umol/l; -30.96 to -5.67), and bone marrow more suppressed (leucopoenia RR 2.02; 1.12 to 3.66, thrombocytopenia RR 6.97; 2.97 to 16.36, anaemia RR 1.67; 1.27 to 2.20). When TOR-I replaced antimetabolites (11 trials, 3966 participants), AR and CMV were reduced (RR 0.84; 0.71 to 0.99 and RR 0.49; 0.37 to 0.65) but hypercholesterolaemia was increased (RR 1.65; 1.32 to 2.06). When low was compared to high-dose TOR-I, with equal CNI dose (10 trials, 3175 participants), AR was increased (RR 1.23; 1.06 to 1.43) but GFR higher (WMD 4.27 ml/min; 1.12 to 7.41). When low-dose TOR-I and standard-dose CNI were compared to higher-dose TOR-I and reduced CNI AR was reduced (RR 0.67; 0.52 to 0.88), but GFR also reduced (WMD -9.46 ml/min; -12.16 to -6.76). There was no significant difference in mortality, graft loss or malignancy risk demonstrated for TOR-I in any comparison. Generally surrogate endpoints for graft survival favoured TOR-I (lower risk of acute rejection and higher GFR) and surrogate endpoints for patient outcomes were worsened by TOR-I (bone marrow suppression, lipid disturbance). Long-term hard-endpoint data from methodologically robust randomised trials are still needed. Monoclonal and polyclonal antibody therapy for treating acute rejection Strategies for treating AR include pulsed steroids, an antibody (Ab) preparation, the alteration of background immunosuppression, or combinations of these options. In 2002, in the USA 61.4% of patients with AR received steroids, 20.4% received Ab and 18.2% received both. The Ab available for AR are not new: horse and rabbit derived polyclonal antibodies (ATG and ALG) have been used for 35 years, and a mouse monoclonal antibody (muromonab-CD3) became available in the late 1980s. These preparations remove the functional T-cell population from circulation, producing powerful saturation immunosuppression which is useful for AR but which may be complicated by immediate toxicity and higher rates of infection and malignancy. The aim of this study was to systematically evaluate and synthesise all evidence available to clinicians for treating AR in kidney recipients. We identified 49 reports from 21 randomised trials involving 1394 participants. Outcome measures were inconsistent and incompletely defined across trials. Fourteen trials (965 patients) compared therapies for 1st AR episodes (8 Ab versus steroid, 2 Ab versus another Ab, 4 other comparisons). In treating first rejection, Ab was better than steroid in reversing AR (RR 0.57; CI 0.38 to 0.87) and preventing graft loss (RR 0.74; CI 0.58 to 0.95) but there was no difference in preventing subsequent rejection (RR 0.67; CI 0.43 to 1.04) or death (RR 1.16; CI 0.57 to 2.33) at 1 year. Seven trials (422 patients) investigated Ab treatment of steroid-resistant rejection (4 Ab vs another Ab, 1 different doses Ab, 1 different formulation Ab, 2 other comparisons). There was no benefit of muromonab-CD3 over ATG or ALG in reversing rejection (RR 1.32; CI 0.33 to 5.28), preventing subsequent rejection (RR 0.99; CI 0.61 to 1.59), graft loss (RR 1.80; CI 0.29 to 11.23) or death (RR 0.39; CI 0.09 to 1.65). Given the clinical problem caused by AR, comparable data are sparse, and clinically important differences in outcomes between widely used interventions have not been excluded. Standardised reproducible outcome criteria are needed. Validity of cancer data in an end stage kidney disease registry Registries vary in whether the data they collect are given voluntarily or as a requirement of law, the completeness of population coverage, the breadth of data collected and whether data are assembled directly or indirectly through linkage to other databases. Data quality is crucial but difficult to measure objectively. Formal audit of ANZDATA cancer records has not previously taken place. The aim of this study was to assess agreement of records of incident cancer diagnoses held in ANZDATA (voluntary reporting system) with those reported under statute to the New South Wales (NSW) state Central Cancer Registry (CCR), to explore the strengths and weaknesses of both reporting systems, and to measure the impact of any disagreement on results of cancer analyses. From 1980-2001, 9453 residents received dialysis or transplantation in NSW. Records from ANZDATA registrants were linked to CCR using probabilistic matching and agreement between registries for patients with 1 or more cancers, all cancers and site-specific cancer was estimated using the kappa-statistic (κ). ANZDATA recorded 867 cancers in 779 (8.2%) registrants; CCR 867 cancers in 788 (8.3%), with κ =0.76. ANZDATA had sensitivity 77.3% (CI 74.2 to 80.2), specificity 98.1% (CI 97.7 to 98.3) if CCR records were regarded as the reference standard. Agreement was similar for diagnoses whilst receiving dialysis (κ =0.78) or after transplantation (κ =0.79), but varied by cancer type. Melanoma (κ =0.61) and myeloma (κ =0.47) were less good; lymphoma (κ =0.80), leukaemia (κ =0.86) and breast cancer (κ =0.85) were very good. Artefact accounted for 20.8% non-concordance but error and misclassification did occur in both registries. Cancer risk did not differ in any important way whether estimated using ANZDATA or CCR records. Quality of cancer records in ANZDATA are high, differences largely explicable, and seem unlikely to alter results of analyses. Risk of cancer after kidney transplantation Existing data on the magnitude of excess risk of cancer across different kidney recipient groups are sparse. Quantifying an individual transplant candidate’s cancer risk informs both pre-transplant counselling, treatment decisions and has implications for monitoring, screening and follow-up after transplantation. The aims of this study were firstly to establish the risk of cancer in the post-transplant population compared to that experienced by the general population, and secondly to quantify how excess risk varied within the transplanted population, seeking to establish meaningful absolute risk estimates for post-transplant cancer based on unalterable recipient characteristics known a priori at the time of transplantation. 15,183 residents of Australia and New Zealand had a transplant between 1963 and 2004, and were followed for a median of 7.2 years (130,186 person-years), with 1642 (10.8%) developing cancer. Overall, kidney recipients had 3 times the cancer risk, with risk inversely related to age (Standardised Incidence Ratio of 15 to 30 in children reducing to 2 in people > 65 years). Female recipients aged 25 -29 had rates of cancer (779.2/100,000) equivalent to women aged 55 - 59 from the general population. The risk pattern of lymphoma, colorectal and breast cancer was similar to the overall age trend, melanoma showed less variability across ages and prostate cancer showed no risk increase. Within the transplanted population cancer risk was affected by age differently for each sex (P=0.007), and was elevated for recipients with prior non-skin malignancy (Hazard Ratio: HR 1.40; 1.03 to 1.89), of white race (HR 1.36; 1.12 to 1.89), but reduced for those with diabetic ESKD (HR 0.67; 0.50 to 0.89) Rates of cancer in kidney recipients were similar to non-transplanted people 20 -30 years older, but risk differed across patient groups. Men aged 45 - 54 at transplantation with graft function at 10 years had a risk of cancer that varied from 1 in 13 (non-white, diabetic ESKD, no prior cancer) to 1 in 5 (white, prior cancer, ESKD from other causes).

PhD
Introduction Kidney transplantation confers both survival and quality of life advantages over dialysis for most people with end-stage kidney disease (ESKD). The mortality rate on dialysis is 10-15% per year, compared with 2-4% per year post-transplantation. Short-term graft survival is related to control of the acute rejection process, requiring on-going immunosuppression. Most current immunosuppressive algorithms include one of the calcineurin inhibitors (CNI: cyclosporin or tacrolimus), an anti-metabolite (azathioprine or mycophenolate) and corticosteroids, with or without antibody induction agents (Ab) given briefly peri-transplantation. Despite this approach, between 15-35% of recipients undergo treatment for an episode of acute rejection (AR) within one year of transplantation. Transplantation is not without risk, and relative mortality rates for kidney recipients after the first post-transplant year remain 4-6 times that of the general population. Longer-term transplant and recipient survival are related to control of chronic allograft nephropathy (rooted in the interplay of AR, non-immunological factors, and the chronic nephrotoxicity of CNI) and limitation of the complications of chronic ESKD and long-term immunosuppression: cardiovascular disease, cancer and infection, which are responsible for 22%, 39% and 21% of deaths respectively. This thesis is presented as published works on the theme of immunosuppression and cancer after kidney transplantation. The work presented in the first chapters of this thesis has striven to identify, evaluate, synthesise and distil the entirety of evidence available of new and established immunosuppressive drug agents through systematic review of randomised trial data, with particular emphasis on quantifying harms of treatment. The final chapters use inception cohort data from the Australian and New Zealand Dialysis and Transplant Registry (ANZDATA), which is first validated then used to explore the risk of cancer in more detail than was possible from trial data alone. Interleukin 2 receptor antagonists Interleukin-2 receptor antagonists (IL2Ra, commercially available as basiliximab and daclizumab) are humanised or chimeric IgG monoclonal antibodies to the alpha subunit of the IL2 receptor present only on activated T lymphocytes, and the rationale for their use has been as induction agents peri-transplantation. Introduced in the mid-1990s, IL2Ra use has increased globally, and by 2003 38% of new kidney transplant recipients in the United States and 25% in Australasia received an IL2Ra. This study aimed to systematically identify and synthesise the evidence of effects of IL2Ra as an addition to standard therapy, or as an alternative to other induction agents. We identified 117 reports from 38 randomised trials involving 4893 participants. Where IL2Ra were compared with placebo (17 trials; 2786 patients), graft loss was not different at one (Relative Risk -RR 0.84; 0.64 to 1.10) or 3 years (RR 1.08; 0.71 to1.64). AR was reduced at 6 months (RR 0.66; 0.59 to 0.74) and at 1 year (RR 0.66; 0.59 to 0.74) but cytomegalovirus (CMV) disease (RR 0.82; CI 0.65 to 1.03) and malignancy (RR 0.67; 0.33 to1.36) were not different. Where IL2Ra were compared with other antibody therapy no significant differences in treatment effects were demonstrated, but IL2Ra had significantly fewer side effects. Given a 40% risk of rejection, 7 patients would need treatment with IL2Ra in addition to standard therapy, to prevent 1 patient having rejection, with no definite improvement in graft or patient survival. There was no apparent difference between basiliximab and daclizumab. Tacrolimus versus cyclosporin for primary immunosuppression There are pronounced global differences in CNI use; 63% of new kidney transplant recipients in the USA but only 22% in Australia receive tacrolimus as part of the initial immunosuppressive regimen. The side effects of CNI differ: tacrolimus is associated more with diabetes and neurotoxicity, but less with hypertension and dyslipidaemia than cyclosporin, with uncertainty about equivalence of nephrotoxicity or how these relate to patient and graft survival, or impact on patient compliance and quality of life. This study aimed to systematically review and synthesise the positive and negative effects of tacrolimus and cyclosporin as initial therapy for renal transplant recipients. We identified 123 reports from 30 randomised trials involving 4102 participants. At 6 months graft loss was reduced in tacrolimus-treated recipients (RR 0•56; 0•36 to 0•86), and this effect persisted for 3 years. The relative reduction in graft loss with tacrolimus diminished with higher levels of tacrolimus (P=0.04), but did not vary with cyclosporin formulation (P=0.97) or cyclosporin level (P=0.38). At 1 year, tacrolimus patients suffered less AR (RR 0•69; 0•60 to 0•79), and less steroid-resistant AR (RR 0•49; 0•37 to 0•64), but more insulin-requiring diabetes (RR 1•86; 1•11 to 3•09), tremor, headache, diarrhoea, dyspepsia and vomiting. The relative excess in diabetes increased with higher levels of tacrolimus (P=0.003). Cyclosporin-treated recipients experienced significantly more constipation and cosmetic side-effects. We demonstrated no differences in infection or malignancy. Treating 100 recipients with tacrolimus instead of cyclosporin for the 1st year post-transplantation avoids 12 suffering acute rejection and 2 losing their graft but causes an extra 5 to become insulin dependent diabetics, thus optimal drug choice may vary among patients. Target of rapamycin inhibitors for primary immunosuppression Target of rapamycin inhibitors (TOR-I) are among the newest immunosuppressive agents and have a novel mode of action but uncertain clinical role. Sirolimus is a macrocyclic lactone antibiotic and everolimus is a derivative of sirolimus. Both prevent DNA synthesis resulting in arrest of the cell cycle. Animal models suggested TOR-I would provide synergistic immunosuppression when combined with CNI, but early clinical studies demonstrated synergistic nephrotoxicity. Since then diverse trials have explored strategies that avoid this interaction and investigated other potential benefits. The aim of this study was to systematically identify and synthesise available evidence of sirolimus and everolimus when used in initial immunosuppressive regimens for kidney recipients. We identified 142 reports from 33 randomised trials involving 7114 participants, with TOR-I evaluated in four different primary immunosuppressive algorithms: as replacement for CNI, as replacement for antimetabolites, in combination with CNI at low and high dose, and with variable dose of CNI. When TOR-I replaced CNI (8 trials, 750 participants), there was no difference in AR (RR 1.03; 0.74 to 1.44), but creatinine was lower (WMD -18.31 umol/l; -30.96 to -5.67), and bone marrow more suppressed (leucopoenia RR 2.02; 1.12 to 3.66, thrombocytopenia RR 6.97; 2.97 to 16.36, anaemia RR 1.67; 1.27 to 2.20). When TOR-I replaced antimetabolites (11 trials, 3966 participants), AR and CMV were reduced (RR 0.84; 0.71 to 0.99 and RR 0.49; 0.37 to 0.65) but hypercholesterolaemia was increased (RR 1.65; 1.32 to 2.06). When low was compared to high-dose TOR-I, with equal CNI dose (10 trials, 3175 participants), AR was increased (RR 1.23; 1.06 to 1.43) but GFR higher (WMD 4.27 ml/min; 1.12 to 7.41). When low-dose TOR-I and standard-dose CNI were compared to higher-dose TOR-I and reduced CNI AR was reduced (RR 0.67; 0.52 to 0.88), but GFR also reduced (WMD -9.46 ml/min; -12.16 to -6.76). There was no significant difference in mortality, graft loss or malignancy risk demonstrated for TOR-I in any comparison. Generally surrogate endpoints for graft survival favoured TOR-I (lower risk of acute rejection and higher GFR) and surrogate endpoints for patient outcomes were worsened by TOR-I (bone marrow suppression, lipid disturbance). Long-term hard-endpoint data from methodologically robust randomised trials are still needed. Monoclonal and polyclonal antibody therapy for treating acute rejection Strategies for treating AR include pulsed steroids, an antibody (Ab) preparation, the alteration of background immunosuppression, or combinations of these options. In 2002, in the USA 61.4% of patients with AR received steroids, 20.4% received Ab and 18.2% received both. The Ab available for AR are not new: horse and rabbit derived polyclonal antibodies (ATG and ALG) have been used for 35 years, and a mouse monoclonal antibody (muromonab-CD3) became available in the late 1980s. These preparations remove the functional T-cell population from circulation, producing powerful saturation immunosuppression which is useful for AR but which may be complicated by immediate toxicity and higher rates of infection and malignancy. The aim of this study was to systematically evaluate and synthesise all evidence available to clinicians for treating AR in kidney recipients. We identified 49 reports from 21 randomised trials involving 1394 participants. Outcome measures were inconsistent and incompletely defined across trials. Fourteen trials (965 patients) compared therapies for 1st AR episodes (8 Ab versus steroid, 2 Ab versus another Ab, 4 other comparisons). In treating first rejection, Ab was better than steroid in reversing AR (RR 0.57; CI 0.38 to 0.87) and preventing graft loss (RR 0.74; CI 0.58 to 0.95) but there was no difference in preventing subsequent rejection (RR 0.67; CI 0.43 to 1.04) or death (RR 1.16; CI 0.57 to 2.33) at 1 year. Seven trials (422 patients) investigated Ab treatment of steroid-resistant rejection (4 Ab vs another Ab, 1 different doses Ab, 1 different formulation Ab, 2 other comparisons). There was no benefit of muromonab-CD3 over ATG or ALG in reversing rejection (RR 1.32; CI 0.33 to 5.28), preventing subsequent rejection (RR 0.99; CI 0.61 to 1.59), graft loss (RR 1.80; CI 0.29 to 11.23) or death (RR 0.39; CI 0.09 to 1.65). Given the clinical problem caused by AR, comparable data are sparse, and clinically important differences in outcomes between widely used interventions have not been excluded. Standardised reproducible outcome criteria are needed. Validity of cancer data in an end stage kidney disease registry Registries vary in whether the data they collect are given voluntarily or as a requirement of law, the completeness of population coverage, the breadth of data collected and whether data are assembled directly or indirectly through linkage to other databases. Data quality is crucial but difficult to measure objectively. Formal audit of ANZDATA cancer records has not previously taken place. The aim of this study was to assess agreement of records of incident cancer diagnoses held in ANZDATA (voluntary reporting system) with those reported under statute to the New South Wales (NSW) state Central Cancer Registry (CCR), to explore the strengths and weaknesses of both reporting systems, and to measure the impact of any disagreement on results of cancer analyses. From 1980-2001, 9453 residents received dialysis or transplantation in NSW. Records from ANZDATA registrants were linked to CCR using probabilistic matching and agreement between registries for patients with 1 or more cancers, all cancers and site-specific cancer was estimated using the kappa-statistic (κ). ANZDATA recorded 867 cancers in 779 (8.2%) registrants; CCR 867 cancers in 788 (8.3%), with κ =0.76. ANZDATA had sensitivity 77.3% (CI 74.2 to 80.2), specificity 98.1% (CI 97.7 to 98.3) if CCR records were regarded as the reference standard. Agreement was similar for diagnoses whilst receiving dialysis (κ =0.78) or after transplantation (κ =0.79), but varied by cancer type. Melanoma (κ =0.61) and myeloma (κ =0.47) were less good; lymphoma (κ =0.80), leukaemia (κ =0.86) and breast cancer (κ =0.85) were very good. Artefact accounted for 20.8% non-concordance but error and misclassification did occur in both registries. Cancer risk did not differ in any important way whether estimated using ANZDATA or CCR records. Quality of cancer records in ANZDATA are high, differences largely explicable, and seem unlikely to alter results of analyses. Risk of cancer after kidney transplantation Existing data on the magnitude of excess risk of cancer across different kidney recipient groups are sparse. Quantifying an individual transplant candidate’s cancer risk informs both pre-transplant counselling, treatment decisions and has implications for monitoring, screening and follow-up after transplantation. The aims of this study were firstly to establish the risk of cancer in the post-transplant population compared to that experienced by the general population, and secondly to quantify how excess risk varied within the transplanted population, seeking to establish meaningful absolute risk estimates for post-transplant cancer based on unalterable recipient characteristics known a priori at the time of transplantation. 15,183 residents of Australia and New Zealand had a transplant between 1963 and 2004, and were followed for a median of 7.2 years (130,186 person-years), with 1642 (10.8%) developing cancer. Overall, kidney recipients had 3 times the cancer risk, with risk inversely related to age (Standardised Incidence Ratio of 15 to 30 in children reducing to 2 in people > 65 years). Female recipients aged 25 -29 had rates of cancer (779.2/100,000) equivalent to women aged 55 - 59 from the general population. The risk pattern of lymphoma, colorectal and breast cancer was similar to the overall age trend, melanoma showed less variability across ages and prostate cancer showed no risk increase. Within the transplanted population cancer risk was affected by age differently for each sex (P=0.007), and was elevated for recipients with prior non-skin malignancy (Hazard Ratio: HR 1.40; 1.03 to 1.89), of white race (HR 1.36; 1.12 to 1.89), but reduced for those with diabetic ESKD (HR 0.67; 0.50 to 0.89) Rates of cancer in kidney recipients were similar to non-transplanted people 20 -30 years older, but risk differed across patient groups. Men aged 45 - 54 at transplantation with graft function at 10 years had a risk of cancer that varied from 1 in 13 (non-white, diabetic ESKD, no prior cancer) to 1 in 5 (white, prior cancer, ESKD from other causes).

Après ingestion orale d'aliments contenant le parasite responsable de la toxoplasmose, T. Gondii traverse la barrière intestinale et se dissémine rapidement dans tout l'organisme. Nous avons étudié la réponse immunitaire induite dans le poumon dans un modèle de souris C57BL/6 sensibles à la phase aigue͏̈ d'infection. Nos résultats montrent une inflammation pulmonaire interstitielle in situ à J11 post infection. Les lymphocytes T CD4+ et CD8+ activés ont un phénotype de cellules apoptotiques ex vivo et sont en état d'anergie réversible par de l'IL-2 exogène après re-stimulation in vitro. De plus, nous avons mis en évidence l'expansion de cellules CD11b+ et GR1+ inhibant la prolifération de lymphocytes au travers de la synthèse de NO. Nous pouvons conclure que l'infection aigue͏̈ par T. Gondii induit dans le poumon une pathologie sévère qui conduirait à l'inhibition de la réponse anti-toxoplasmique, et donc favoriserait indirectement la survie et la dissémination du parasite dans l'organisme.

Irradiation with ultraviolet-B light (UV-B) suppresses some cell-mediated immune responses to a variety of antigens, including contact sensitizers. Following UV irradiation there is a modulation of Langerhans cell markers, and keratinocytes are induced to synthesize and secrete tumour necrosis factor-alpha (TNF-α). It has been postulated that there is a photoreceptor in the skin which mediates the effects of UV-B radiation on the immune system. One candidate is urocanic acid (UCA) found naturally in the stratum corneum of the epidermis as the trans-isomer, which converts to the cis-isomer on irradiation. Cis-UCA has been demonstrated to suppress immune reponses in several experimental systems. The mechanism of UV-B induced suppression of contact hypersensitivity (CH) responses and the role of cis-UCA were examined using a murine model of CH. UV-B irradiation was demonstrated to suppress the induction of CH response whilst cis-UCA had little, if any, effect. Next, the migration of dendritic cells (DC) to draining lymph nodes (DLN) following UV-B irradiation or epicutaneous application of UCA isomers was examined in unsensitized mice and mice sensitized with FITC. It was found that UV-B irradiation alone induced DC migration to DLN with a maximum number of DC being present in DLN 48 h following irradiation. In addition UV-B irradiation followed by skin sensitization at the same site enhanced DC migration. In sensitized mice, the percentage of DC bearing FITC and the quantity of FITC per DC were unaltered by prior UV exposure. Further, the percentage of DC expressing Ia or ICAM-1 molecules and the amount of Ia or ICAM-1 expressed per DC was unaffected by UV-B irradiation. In contrast to these results with UV-B irradiation, neither isomer of UCA had any significant effect on DC numbers in DLN in sensitized or unsensitized mice.

L'acide 3(1h-imidazol-4-yl)-propenoique ou acide urocanique est un metabolite de l'histidine present au niveau de la peau et excrete dans la sueur. L'acide urocanique est considere comme un photoprotecteur naturel. De plus, il a ete montre que l'acide urocanique (z) possede une activite dans le domaine de l'immunosuppression, activite dont le mecanisme n'est pas encore connu. Ces observations nous ont conduit a synthetiser des derives de l'acide urocanique dans un double but: ameliorer la solubilite pour rendre la formulation plus aisee, realiser des etudes de relation structure-activite en immunologie. La grande versatilite de l'acide urocanique (tautomerie possible entre les deux atomes d'azote, isomerisation eventuelle, reactivite concurrente des sites basiques, etc. . . ) rend cette molecule difficile a maitriser quand on s'attache a realiser des modulations selectives. Dans un premier temps, des esters de l'acide urocanique ont ete synthetises ce qui nous a amene a envisager diverses methodes d'esterification de facon a mettre au point un processus operatoire permettant d'obtenir plus aisement les urocanates d'alkyle. L'isomerisation photochimique ainsi que la reactivite dienophile des esters prepares ont ete etudiees. Une autre modulation de la structure peut etre obtenue par n-substitution, une telle modification supprimant pour les isomeres (z) des esters urocaniques toute possibilite de liaison intramoleculaire, pourrait avoir des consequences au niveau de l'activite biologique. Nous avons pu realiser regioselectivement cette reaction sur chacun de deux sites possibles par des groupements methyle ou allyle, ce qui a donne acces a des nouveaux produits, de structure non ambigue, determinee par une etude en resonance magnetique nucleaire du #1h et du #1#3c. De plus des differences de conformation entre les isomeres (e) et (z) ont pu etre mises en evidence. Enfin la reactivite des derives n-allyles vis-a-vis des reactions de transposition a ete etudiee, une enamine particulierement stable a pu etre isolee

Macrophages (Mφ) are a major immune cell involved in anti-tumor responses. Mφ activities such as tumor cytotoxicity. presentation of tumor-associated antigens, and stimulation of anti-tumor lymphocytes are all involved in the battle against tumor growth. However, other Mφ activities such as cell growth promotion, angiogenesis, and suppression of anti-tumor lymphocytes aid in tumor growth. This dissertation discusses how tumors control Mφ activities to create favorable environments for tumor growth. Assessment of tumor- and Mφ-derived molecules has enabled me to design models of communication between tumors, Mφ, and other immune cells. A major research focus was to determine how tumor-derived molecules induce Mφ suppressor activity and control Mφ cytotoxicity. Tumor growth induced Mφ to suppress T lymphocyte proliferation by increasing Mφ production of the suppressor molecules prostaglandin E₂ (PGE₂), nitric oxide (NO), and tumor necrosis factor-α (TNF-α). A major finding was that TNF-α's normal up-regulatory action on T-cell proliferation switched to a suppressor action when Mφ were present. The autocrine action of increased TNF-α levels during tumor growth stimulated Mφ PGE₂ and NO synthesis, which suppressed T-cell proliferation.
Ph. D.

Exposure to ultraviolet-B light (UVB) suppresses immune responses to a variety of antigens, including contact sensitizers, in both mice and humans. A number of mediators of UVB-induced immunosuppression have been proposed including DNA damage, cis-urocanic acid, TNF-α and IL-10. UVB irradiation also causes the loss of Langerhans cells (LC) from the skin and the accumulation of LC-derived dendritic cells (CD) in the draining lymph nodes (DLN). A chronic UVB exposure protocol, using broadband (270-350nm) and narrowband (311-312nm) sources, was used to examine the effect of UVB on the skin immune system. Exposure to both sources reduced the numbers of LC in the epidermis. Exposure to broadband but not narrowband UVB induced epidermal thickening in a dose dependent manner. Although damage measured by thickening resulted from broadband UVB only, both sources led to a small increase in sunburn cells, which may represent an apoptotic keratinocyte population. Both sources were equally efficient at inducing the conversation of trans-urocanic acid to the cis-isomer, but only the broad band source suppressed contact hypersensitivity (CH) responses. Thus LC loss and the isomerization of urocanic acid may not be the primary mediators of immunosuppression during chronic UVB exposure. An acute UVB exposure protocol was used to examine the effect of UVB on DC in vivo. Mice were irradiated with an immunosuppressive dose of UVB, and the function and phenotype of DC accumulating in the DLN was examined. Exposure to UVB prior to sensitization did not reduce the ability of DC to induce proliferative responses of hapten sensitized lymph node cells (LNC). The necessary function of DC in mixed lymphocyte reactions was also unaffected by prior exposure to an immunosuppressive dose of UVB. The expression of MHC class II, intercellular adhesion molecule-1 and B7-2 (CD86) on DC was also examined.

The studies presented in this thesis examine the mechanisms of T cell immunosuppression during the graft-versus-host reaction (GVHR). GVHR was induced by the injection of parental lymphoid cells into F1 hybrid recipient mice. The ensuing acute reaction consisted of an initial immunoproliferative phase, followed by the development of profound immunosuppression and histopathological lesions of epithelial and lymphoid tissues. Survivors of the acute reaction developed the persistent immune abnormalities characteristic of chronic GVHR.
We have found that the T cell protein tyrosine kinases p56$ rm sp{lck}$ and p59$ rm sp{fyn}$, involved in signal transduction through the T cell receptor (TCR), are downregulated in the T cells of mice during GVHR. The reduction of lck and fyn was prevented by adrenalectomy of the recipients, and a similar reduction could be induced in normal (non-GVH-reactive) mice by an injection of exogenous cortisone. These findings suggested that the GVHR-induced elevation in endogenous glucocorticoid levels could trigger the decrease of T cell lck and fyn, resulting in a T cell signalling defect during GVHR. In fact, we have demonstrated that glucocorticoids induced a decrease in lck and fyn in T cell clones in vitro. Thus, it appeared that the early GVHR-induced T cell unresponsiveness was due to the glucocorticoid-dependent downregulation of T cell lck and fyn.
We have investigated changes in T cell maturation and selection in the GVHR-dysplastic thymus, which may account for the persistent immune abnormalities of chronic GVHR. Thymocyte TCR expression and usage were aberrant during GVHR; changes included decreased expression of CD3 on CD4$ sp+$8 thymocytes, inconsistent TCR V$ beta$ usage, and appearance of phenotypically autoreactive mature thymocytes. These abnormalities, suggestive of defective positive and negative selection, are likely to result from the GVHR-induced decrease in thymic class II MHC expression. Altered T cell education may lead to the long-term peripheral T cell defects observed in chronic GVHR. Lastly, we report that GVHR-induced cutaneous injury was exacerbated by irradiation of the target tissue, suggesting that in clinical GVHR, irradiation may intensify tissue damage, including thymic epithelial lesions; this could potentially lead to more serious alterations in thymic function, and thus to longer lasting, more severe peripheral T cell immune deficiency.

Ultraviolet radiation (UVR) results in a transient suppression of selected immune responses to antigens encountered following exposure. Urocanic acid (UCA), found in the stratum corneum of the epidermis as the trans-isomer, absorbs UV light whereupon it changes to the cis-isomer. Cis-UCA mimics many of the suppressive effects of UVR on immune responses. Previously cis-UCA has been shown to produce a dose dependent, antigen specific suppression of the delayed hypersensitivity (DH) response to herpes simplex virus (HSV) in a murine model of infection, in an identical manner to UVB-irradiation. A time course of DH revealed an initial response 1 hour after antigen challenge, followed by a second response at 24 hours, both of which were suppressed if the mouse had been UV irradiated or treated with cis-UCA prior to infection. Skin painting murine ears with cis-UCA resulted in a reduction in the number of ATPase positive cells (Langerhans cells) in epidermal sheets prepared 24 hours later, while trans-UCA had no effect. In an attempt to elucidate the mechanism of action of cis-UCA two histamine receptor antagonists were employed; cimetidine (H2) and terfenadine (H1). When cis-UCA was applied together with either of the antagonsists no significant reduction in the number of ATPase positive cells was observed. Similarly if cis-UCA was applied together either antagonists the suppression of the DH response to HSV was blocked. Thus cis-UCA may act through a histamine-like receptor. A number of structural analogues of UCA were tested for their ability to suppress the DH response to HSV to further elucidate the structures required to induce immunosuppression. Tumour necrosis factor-α (TNF-α) has been implicated to be of major importance in the induction of UV-induced immunosuppression. Mice pretreated with neutralizing antibodies to TNF-α were found to be resistant to the immunosuppressive effects of UV, but there was little effect on cis-UCA induced suppression of the DH response to HSV. These results extend the mechanism by which cis-UCA modulates suppression of immune responses.

A functional immune system is a potential barrier to tumor growth and progression. Cancer is caused, in part, by the loss of immune surveillance leading to the inability of the immune system to destroy the cancer cells. Macrophages (Mfs) are essential cellular components of the immune system; they influence immune responses in diverse and fundamental ways. As a consequence, Mfs present targets for tumors to evade, thereby enhancing tumor survival and growth. An interaction between CD40 on Mfs and CD40L on T cells is required for cell-mediated inflammatory responses. The CD40/CD40L interaction is bi-directional; suppressed expression of either protein by the tumor will prevent activation of both Mfs and T cells. We showed that tumor growth suppresses T-cell CD40L expression. Decreased CD40L expression disrupted Mf activation pathways, leading to impaired production of immunostimulatory cytokines, interleukin (IL)-12 and IL-18 by tumor-bearing host (TBH) Mfs. Disruption of CD40L expression, via dysregulation of IL-12 and IL-18 production, impeded T-cell interferon (IFN)-g production, which in turn exacerbated Mf dysfunction. We showed that IFN-g induced interferon consensus sequence binding protein (ICSBP) expression is impaired in TBH Mfs due to tumor cell-derived TGF-b and, to a lesser extent, IL-10. ICSBP induces CD40L, IL-12, and IL-18 expression. Disruption of the CD40/CD40L interaction via lowered CD40L expression generates an immunosuppressive loop that may be a strategy for tumor survival and growth. This was demonstrated by impaired cytotoxicity; via impaired tumor necrosis factor (TNF)-a and nitric oxide (NO) production by TBH Mfs against Meth-KDE tumor cells. Collectively, these studies show that multiple antitumor mechanisms could be enhanced by restoration of CD40L expression.
Master of Science

L'acide docosahexaénoi͏̈que (DHA), un acide gras polyinsaturé de la famille n-3, a été décrit comme capable d'inhiber l'activation du lymphocyte aussi bien in vitro qu'in vivo. Cette inhibition s'accompagne d'une activation de la phospholipase D (PLD). Afin d'élucider les mécanismes mis en jeu lors de l'activation de la PLD par le DHA dans les cellules mononucléées de sang périphérique humain (PBMC), nous avons tout d'abord caractérisé les isoformes de PLD exprimées dans ces cellules ainsi que leur mode de régulation. Les PBMC expriment deux isoformes de PLD, PLD1 et PLD2, aussi bien au niveau des ARN messagers que des protéines, mais sont dépourvues de PLD oléate-sensible. L'activité PLD révélée lors de la stimulation mitogénique après enrichissement en DHA, s'est avérée indépendante des PKC, mais dépendante des petites protéines-G ARF, suggérant une implication de PLD1 principalement. Nous avons également pu mettre en évidence que PLD1 est liée à des structures particulières de la membrane, riches en cholestérol et sphingomyéline, les microdomaines lipidiques ou "rafts". PLD2 en revanche en est exclue. Après enrichissement des membranes cellulaires en DHA et stimulation par la concanavaline A, la PLD1 est délocalisée hors des "rafts", de même que certaines protéines de signalisation telles que les protéines tyrosine kinases Lck ou les protéines ARF. Le traitement des PBMC par des molécules capables de modifier le contenu membranaire des cellules en cholestérol (filipine, méthyl-b-cyclodextrine) ou en sphingomyéline (sphingomyélinase bactérienne), et donc de perturber sélectivement l'organisation des rafts, stimule fortement l'activité PLD et cette activation s'accompagne d'une délocalisation de la protéine PLD1. Parallèlement ces molécules inhibent la réponse lymphoproliférative aux mitogènes. L'ensemble de nos résultats montre que la déstabilisation partielle des microdomaines membranaires provoque la délocalisation de protéines de signalisation ainsi que de la PLD1 hors des microdomaines. La relocalisation de PLD1 dans un environnement lipidique et protéique diffèrent apparaît comme un nouveau mécanisme régulateur, de l'activation de l'enzyme. L'émission d'un signal anti-mitogénique par la PLD pourrait expliquer au moins partiellement l'effet immunosuppresseur du DHA
The inhibition of the lymphoproliferative response by docosahexaenoic acid (DHA, 22:6 n-3) is accompanied by the activation of phospholipase D. To elucidate the mechanisms involved, we characterized the different PLD isoforms of the peripheral blood mononuclear cells (PBMC). These cells express PLD1 and PLD2, both at the m-RNA and protein levels, but they are devoid of oleate-sensitive PLD. PLD activity induced by the mitogenic stimulation of DHA-enriched cells was independent of PKC but dependent of the small G-proteins ARF, indicating that PLD1 was mainly involved. We also pointed out that PLD1 was associated to cholesterol and sphingomyelin-enriched lipid microdomains or "rafts". In contrast, PLD2 was excluded from these structures. After enrichment of the cellular membranes with DHA and stimulation, PLD1 as well as the protein tyrosine kinase Lck and ARF were delocalised out of the rafts. The treatment of cellular membranes by molecules able to modify their cholesterol or shingomyelin contents and to disturb selectively raft organization, strongly stimulated PLD activity and was accompanied by a delocalisation of the PLD1 protein. In parallel, the lymphoproliferative response to mitogenes was decreased. These results show that the partial destabilization of the rafts delocalises signalling proteins and PLD1 out of the rafts. The relocalization of the PLD1 in a different lipid and protein environment appears as a new regulatory mechanisms of PLD activation. Because PLD activation is known to convey antiproliferative signals, this could partially explain the immunosuppression effect of DHA

Thèse doctorat : Biochimie : Villeurbanne, INSA : 200.
En annexe, 1 article extrait de la revue "Journal of biological chemistry, vol. 277, n° 42, 18 oct. 2002, p. 39368-39378" Titre provenant de l'écran-titre. Bibliogr. p. 188-221.

Cette thèse avait pour objectif de fournir des données sur l’efficacité et la tolérance des vaccins dans un contexte d’immunodépression chronique, dans diverses situations cliniques et pour différents antigènes vaccinaux. Un premier essai randomisé chez 95 patients infectés par le VIH a ainsi montré que 15% des patients avaient perdu leurs anticorps protecteurs quatre ans après vaccination contre l��hépatite A, quel que soit le nombre de doses de vaccin reçues dans le schéma de primo-vaccination (2 ou 3 doses). Puis une revue bibliographique systématique et une méta-analyse étendues aux autres antigènes du calendrier vaccinal et à d’autres catégories d’immunodépression (hémodialysés et transplantés d’organe solide) ont confirmé que la durée de persistance des anticorps après vaccination était plus courte chez ces sujets qu’en population générale. La question de la tolérance vaccinale a été abordée au travers de deux études observationnelles : la première n’a pas mis en évidence d’impact significatif des vaccinations sur l’incidence des poussées chez les patients atteint de vascularite systémique, et la seconde a montré que les patients sous corticoïdes rapportaient plus fréquemment des réactions locales modérées ou sévères après vaccination contre la fièvre jaune que les témoins (RR = 8,0 (IC 95% : 1,4 ; 45,9)). Ces différents travaux montrent que la tolérance et l’immunogénicité des vaccins présente des particularités chez les personnes immunodéprimées qui nécessitent d’être prises en compte par la rédaction de recommandations spécifiques en termes de schémas et de fréquence des rappels vaccinaux
Safety, immunogenicity and durability of responses after vaccination are not well documented in the context of immune suppression. We first present the results of a clinical trial in 95 patients living with HIV comparing the immunogenicity of a reinforced 3-dose vaccine scheme against hepatitis A to the standard 2-dose regimen. Maintenance of seroprotection was not improved in the 3-dose arm since 15% of responders had lost protective antibodies by year 4 after immunization in both arms (p=1). We next extended our work to other categories of immune suppression by performing a systematic review of published works providing original long term (i. E. More than 6 months) immunogenicity data with licensed vaccines in three common conditions increasingly met in clinical practice: HIV infection, end-stage renal disease and solid organ transplantation. A meta-analysis of the estimations confirmed that duration of seroprotection provided by most licensed vaccines was shorter in immunocompromised patients than in otherwise healthy persons. Safety issues have been addressed through two observational studies: the first focused on the impact of immunizations on the course of systemic vasculitis and the second showed that patients on corticosteroids reported moderate/severe local reactions more frequently than controls (Relative Risk (95%CI) = 8. 0 (1. 4 to 45. 9)) to the live-attenuated yellow fever vaccine. Safety and immunogenicity of vaccines in persons with impaired immunity are not comparable to that observed in healthy individuals, calling for specific recommendations in these patients