Macrophages (Mφ) have previously been implicated in both acute and chronic renal allograft rejection however the mechanisms remain unclear. In this thesis I set out to explore the effect of the Mφ on the endothelium in the context of renal graft rejection. Initial studies focussed upon human renal allograft tissue from transplant nephrectomies performed because of chronic allograft nephropathy (CAN). Immunostaining was carried out on these tissues (n=29) and control kidney tissue obtained from nephrectomies performed for renal cell carcinoma (n=19). An increased interstitial Mφ infiltrate was found compared to control tissue. Immunostaining for the T cell marker CD3 and the B cell marker CD20 demonstrated that both lymphocyte populations were present in the CAN tissue with almost negligible numbers seen in control tissue. Previous work in the group had demonstrated a reduced number of CD31 positive peritubular capillaries in the tissues used in these studies. In the work undertaken in this thesis, additional analysis was performed to study lymphatic vessels. Immunostaining of control tissue with the lymphatic endothelial cell (LEC) marker podoplanin demonstrated a normal distribution of lymphatic vessels around large interlobular arteries. CAN tissue, however, exhibited an increased lymphatic density with lymphatic vessels evident within the interstitium; a finding verified with two additional LEC markers (LYVE-1 and VEGFR-3). Further investigations examined possible mediators that could be responsible for the reduced microvascular peritubular capillary network and increased lymphatic vessels present in tissues affected by CAN. Previous work had implicated nitric oxide (NO) generated by the enzyme inducible nitric oxide synthase (iNOS) in cardiac allograft rejection. Double immunolabelling for iNOS and the Mφ marker CD68 revealed evidence of Mφ expression of iNOS. No obvious reduction in vascular endothelial growth factor (VEGF)-A was evident although marked expression of VEGF-A was found in CD20 positive B cells within CAN tissue. Occasional interstitial cells expressed the lymphangiogenic growth factor VEGF-C, with double labelling studies indicating occasional CD68 +ve Mø that were positive for VEGF-C. In vitro studies were undertaken to dissect the interaction between Mø and microvascular endothelial cells (MCEC-1) using well established in vitro co-culture techniques. Co-culture of cytokine activated bone marrow derived Mø with MCEC-1 cells (a murine cardiac microvascular endothelial cell line) resulted in increasing levels of MCEC-1 apoptosis and a reduced cell number over a 24-hour time course. Non-activated Mø or cytokines alone were not cytotoxic. Co-cultures were performed in the presence of L-Nimino- ethyl lysine (L-Nil), a specific inhibitor of iNOS (control D-N6- (1-iminoethyl)-lysine (D-Nil)). L-Nil significantly inhibited MCEC-1 apoptosis and preserved cell number implicating a major role for NO in Mø-mediated MCEC-1 death. Importantly, L-Nil treatment did not affect TNFα production by cytokines suggesting that TNFα is not involved in MCEC-1 death in this in vitro experimental system. Experiments were then undertaken involving the depletion of Mø in a murine model of acute renal allograft rejection. Renal transplants were performed between donor Balb/c mice and either FVB/N CD11b-DTR mice transgenic for the diphtheria toxin receptor (DTR) under the CD11b promoter or control non-transgenic FVB/N mice. Diphtheria toxin (DT) was administered on days 3 and 5 to induce Mø depletion and mice sacrificed at day 7. Isograft controls were also performed between FVB/N mice. Murine allografts exhibited marked interstitial F4/80 positive Mø infiltration with expression of iNOS in the allografts. There was significant loss of peritubular capillaries (PTC) in allografts compared to isografts, indicating microvascular injury. DT treated CD11b-DTR mice exhibited 75% reduction in Mø infiltration and this was associated with dramatic microvascular protection. B and T cells were not evident in the isograft but significant accumulation of B and T cells was present in the allograft and not affect by Mø depletion. Interestingly, there was an increase in the number of podoplanin positive lymphatic vessels in the allograft compared to the isograft, which was significantly inhibited following Mø depletion. The final area of study focussed upon attempts to isolate lymphatic endothelial cells in vitro. Two types of vascular cells (HUVECs and HDMECs) were analysed by flow cytometry for LEC markers and immunofluorescence to phenotype the cells. Magnetic bead sorting was then undertaken to isolate discrete populations of endothelial cells expressing LEC markers. The murine studies reinforce the cytotoxic potential of Mø and supports a role for Mø in the deleterious rarefaction of microvascular interstitial vessels with resultant tissue hypoxia and ischaemia. Furthermore, these data support the involvement of Mø in the interstitial lymphangiogenesis that may occur in renal allografts. Furthermore, the study of human allograft tissue indicates that microvascular rarefaction and an increase in intrarenal lymphatic vessels occurs in human disease. Lastly, Mø expression of iNOS and VEGF-C suggests that Mø are involved in key processes that may adversely affect graft outcome.

No description available.

The natural history for patients with de novo donor-specific antibodies (dnDSA) and the risk factors for its development have not been well defined. Furthermore, clinical and histologic correlation with serologic data is limited. We studied 315 consecutive renal transplants without pre-transplant donor-specific antibody (DSA), with a mean follow-up of 6.2 ± 2.9 years. Protocol (n = 215) and for cause (n = 163) biopsies were analyzed. Solid phase assays were used to screen for dnDSA post-transplant. A total of 47 out of 315 (15%) patients developed dnDSA at a mean of 4.6 ± 3.0 years post-transplant. Independent predictors of dnDSA were HLA-DRβ1 MM > 0 (OR 5.66, p < 0.006); and non-adherence (OR 8.75, p < 0.001); with a strong trend toward clinical rejection episodes preceding dnDSA (OR 1.57 per rejection episode, p=0.061). The median 10-year graft survival for those with dnDSA was lower than the No dnDSA group (57% vs. 96%, p < 0.0001). Pathology consistent with antibody-mediated injury occurred and progressed in patients with dnDSA in the absence of graft dysfunction. Furthermore, non-adherence and cellular rejection contributed to both dnDSA development and the risk of progression to graft loss. (Human leukocyte antigen) HLA epitope matching is a novel strategy that may minimize dnDSA development. HLAMatchmaker software was used to characterize epitope mismatches at 395 potential HLA-DR/DQ/DP conformational epitopes for a subset of 286 donor–recipient pairs in which samples were available for high-resolution HLA-typing. Epitope specificities were assigned using single antigen HLA bead analysis and correlated with known monoclonal alloantibody epitope targets. Locus-specific epitope mismatches were more numerous in patients who developed HLA-DR dnDSA alone (21.4 vs. 13.2, p<0.02) or HLA-DQ dnDSA alone (27.5 vs. 17.3, p<0.001). An optimal threshold for epitope mismatch (10 for HLA-DR, 17 for HLA-DQ) was defined that was associated with minimal development of Class II dnDSA using a receiver operating characteristic analysis. Applying these thresholds, 0% and 2.7% of patients developed dnDSA against HLA-DR and HLA-DQ, respectively, after a median of 6.9 years follow-up. Epitope specificity analysis revealed that 3 HLA-DR and 3 HLA-DQ epitopes were independent multivariate predictors of Class II dnDSA when mismatched between the donor and recipient. HLA-DR and DQ epitope matching outperforms traditional low-resolution antigen-based matching and has the potential to minimize the risk of de novo Class II DSA development, thereby improving long-term graft outcome.

Solid organ transplantation is the gold standard treatment for a variety of conditions that result in organ failure. However, despite considerable advances in clinical transplantation in recent decades, the almost ubiquitous requirement of life-long immunosuppression of transplant recipients persists and is complicated by graft loss to rejection in the long term and multiple serious adverse effects that are frequently life limiting. Helminths currently infect more than one quarter of the world’s population and it is now well established that their success as parasites is the result of active immunomodulation of the host immune response. Whilst this primarily secures ongoing survival of the parasites, in some cases helminth-induced immunomodulation can be beneficial to the infected host and is not associated with the adverse sequelae of pharmacological immunosuppression. An emerging body of evidence suggests that harmful immune responses to alloantigens can be suppressed by helminths, but little mechanistic data exists and the active immunomodulators involved have remained hitherto unidentified. The hypothesis behind this thesis is that the model intestinal nematode, Heligmosomoides polygyrus, produces immunomodulatory molecules that can suppress responses to allo- and auto-antigens in animal models of transplantation and autoimmunity, and that some of these molecules could potentially be exploited as novel therapeutic agents. Full-thickness skin grafting was performed between fully-allogeneic mouse strains (BALB/c to C57BL/6). Recipient mice infected with H. polygyrus immediately prior to transplantation showed significantly prolonged allograft survival. Likewise, protection from allograft rejection could be replicated in recipient mice in which H. polygyrus excretory-secretory products (HES) (isolated from culture of adult worms) were delivered by continuous infusion via surgically implanted osmotic minipumps. A number of potential mechanisms underlying allograft protection were identified including induction of CD4+CD25+Foxp3+ regulatory T cells (Treg) and suppression of Th1 and Th17 effector CD4+ T cell phenotypes. H. polygyrus and HES were further shown to ameliorate disease in murine (pMOG) experimental autoimmune encephalomyelitis and colitis induced by T cell transfer. In addition to expansion of Treg, H. polygyrus-mediated protection against EAE was found to be almost completely lost in IL-4 receptor deficient mice, indicating a protective role of Th2 immune responses in this context. Finally, the mechanisms of action of the newly-identified TGF-β mimic, TGM, contained within HES were investigated. Despite bearing no sequence homology or structural resemblance to TGF-β, TGM was shown to act through the TGF-β receptor complex to induce Treg in human and mouse CD4+ T cells in vitro and to suppress murine allogeneic skin graft rejection in vivo. TGM may represent the origin of a safe, effective and long-overdue novel alternative to current immunosuppression therapy.

No description available.

El Tumor de Células Gigantes es un tumor de comportamiento localmente agresivo y que asienta a nivel óseo próximo a las articulaciones en pacientes jóvenes. La larga trayectoria en el tratamiento de los tumores óseos en el Hospital Universitario Virgen de la Arrixaca y la controversia actual en el diagnóstico y tratamiento de este tumor nos llevó a realizar esta Memoria. Todos los pacientes de la serie fueron sometidos a una completa anamnesis y exploración física y se cuantificaron los intervalos transcurridos desde la aparición de los síntomas, momento de consulta, diagnóstico y tratamiento. Se valora las técnicas diagnósticas empleadas, así como el tratamiento quirúrgico bien mediante procedimiento intralesional o resección amplia y las técnicas de reconstrucción. Se estudia la integración de los aloinjertos estructurales mediante la escala ISOLS y la integración del aloinjerto triturado mediante la escala VIIT. También se valora los resultados funcionales mediante la escala EFCOM. / Giant cell tumor is a locally aggressive bone tumor and it is located in the end of the long bones near of joints in young patients. The long history in the treatment of bone tumors at the Hospital Universitario Virgen de la Arrixaca and the current controversy in the diagnosis and treatment of this tumor led us to this report. All patients in the series underwent a complete history and physical examination and quantified the interval since the onset of symptoms, time of consultation, diagnosis and treatment. It assesses the diagnostic techniques used and the surgical procedure or by intralesional or wide resection and reconstruction techniques. We study the integration of structural allografts by ISOLS scale and integration of crushed allografts by the VIIT scale. It also assesses the functional results by EFCOM scale.

(ENGLISH) Background: The investigation of the immunosuppression usage in cardiovascular surgery and interventional cardiology is, at present, concentrated on three main topics: 1) influence on intimal hyperplasia of coronary and peripheral vascular reconstructions 2) influence on rejection of allogeneic vascular grafts and 3) influence on intimal hyperplasia of coronary arteries after endovascular interventions. Modern immunosuppressive drug FK506 (Tacrolimus) could have a positive effect for these indications. In experimental study, FK506 inhibited rejection of arterial allografts and also inhibited intimal hyperplasia in percutaneous coronary interventions. Aims: The purpose of this study was to evaluate the effect of systemic tacrolimus treatment on the process of arterialisation of allogeneic and syngeneic venous grafts in a rat vein-to-artery implantation model. Material and Methods: Lewis (LEW) rats were used as recipients of syngeneic (Lewis) or allogeneic (Brown-Norway; BN) iliolumbar veins which were implanted into abdominal aorta. Recipients were divided into six groups. In groups A, E and F were animals after syngeneic (LEW to LEW) and in groups B, C and D were animals after allogeneic (BN to LEW) transplantations. Animals in the groups C and F had daily intramuscular injections of...

(ENGLISH) Background: The investigation of the immunosuppression usage in cardiovascular surgery and interventional cardiology is, at present, concentrated on three main topics: 1) influence on intimal hyperplasia of coronary and peripheral vascular reconstructions 2) influence on rejection of allogeneic vascular grafts and 3) influence on intimal hyperplasia of coronary arteries after endovascular interventions. Modern immunosuppressive drug FK506 (Tacrolimus) could have a positive effect for these indications. In experimental study, FK506 inhibited rejection of arterial allografts and also inhibited intimal hyperplasia in percutaneous coronary interventions. Aims: The purpose of this study was to evaluate the effect of systemic tacrolimus treatment on the process of arterialisation of allogeneic and syngeneic venous grafts in a rat vein-to-artery implantation model. Material and Methods: Lewis (LEW) rats were used as recipients of syngeneic (Lewis) or allogeneic (Brown-Norway; BN) iliolumbar veins which were implanted into abdominal aorta. Recipients were divided into six groups. In groups A, E and F were animals after syngeneic (LEW to LEW) and in groups B, C and D were animals after allogeneic (BN to LEW) transplantations. Animals in the groups C and F had daily intramuscular injections of...

Human leukocyte antigen-G (HLA-G), a non-classical MHC I protein, plays an essential role in immune tolerance and is associated with a lower incidence of graft rejection and cardiac allograft vasculopathy (CAV). To examine the pattern of HLA-G expression post-transplantation we determined that HLA-G can be up-regulated in smooth muscle cells (SMCs) following exposure to everolimus. We also determined that HLA-G at 500 and 1000 ng/ml reduces SMC proliferation. In further studies, treatment with HLA-G inhibited TNFα-stimulated neutrophil adhesion to endothelial cells (ECs) at all concentrations tested (0.1-1 ng/ml), suggesting a role in inflammation. The expression of HLA-G is influenced by a polymorphism in the HLA-G gene. We sought to determine if the 14bp insertion/deletion polymorphism can predict the development of CAV. There was no association between this polymorphism and CAV; however, this study had a small number of patients; therefore further investigations are needed to confirm these findings.

No description available.