Treatment-dependent loss of polyfunctional CD8+ T-cell responses in HIV-infected kidney transplant recipients is associated with herpesvirus reactivation.

Antiretroviral-therapy has dramatically changed the course of HIV infection and HIV-infected (HIV(+)) individuals are becoming more frequently eligible for solid-organ transplantation. However, only scarce data are available on how immunosuppressive (IS) strategies relate to transplantation outcome and immune function. We determined the impact of transplantation and immune-depleting treatment on CD4+ T-cell counts, HIV-, EBV-, and Cytomegalovirus (CMV)-viral loads and virus-specific T-cell immunity in a 1-year prospective cohort of 27 HIV(+) kidney transplant recipients. While the results show an increasing breadth and magnitude of the herpesvirus-specific cytotoxic T-cell (CTL) response over-time, they also revealed a significant depletion of polyfunctional virus-specific CTL in individuals receiving thymoglobulin as a lymphocyte-depleting treatment. The disappearance of polyfunctional CTL was accompanied by virologic EBV-reactivation events, directly linking the absence of specific polyfunctional CTL to viral reactivation. The data provide first insights into the immune-reserve in HIV+ infected transplant recipients and highlight new immunological effects of thymoglobulin treatment. Long-term studies will be needed to assess the clinical risk associated with thymoglobulin treatment, in particular with regards to EBV-associated lymphoproliferative diseases.

Graft versus host disease in a rat small bowel transplant model after T-cell depleted donor specific bone marrow infusion

Baixas doses de irradiação associadas à infusão de células da medula óssea não previnem a ocorrência da reação do enxerto versus hospedeiro após o transplante intestinal. OBJETIVO: Neste estudo foi avaliado a potencial vantagem em estender o regime imunossupressor associado a infusão de células de medula óssea do doador depletadas de células T na prevenção da reação do enxerto versus hospedeiro após o transplante intestinal. MÉTODOS: Transplante heterotópico de intestino delgado foi realizado em ratos Lewis como receptores e da como doadores, distribuídos em cinco grupos de acordo com a duração da imunossupressão, irradiação e do uso de medula óssea normal ou depletada: G1 (n=6), sem irradiação e G2 (n=9), G3 (n=4), G4 (n=5) e G5 (n=6) foram irradiados com 250 rd. Grupos1, 2, 4 e G3 e 5 foram infundidos com 100 x 10(6) células da medula normal e depletada respectivamente. Animais no G1,2,3 foram imunossuprimidos com 1mg/kg/FK506/ IM por cinco dias e G4 e cinco por 15 dias. Anticorpos monoclonais contra células CD3 e colunas magnéticas foram utilizadas para a depleção da medula óssea. Os animais foram examinados para a presença de rejeição, reação do enxerto versus hospedeiro, chimerismo e biópsias intestinais e da pele. RESULTADOS: Rejeição mínima foi observada em todos os grupos; entretanto, a reação do enxerto versus hospedeiro somente nos animais irradiados. Extensão da imunossupressão alterou a gravidade da reação nos animais dos G4 e 5. Rejeição foi a causa mortis no G1 e a reação do enxerto versus hospedeiro nos Grupos 2,3,4 e 5, não controlada com a infusão de medula óssea depletada. O chimerismo total e de células T do doador foi estatisticamente maior nos grupos irradiados em comparação ao G1. CONCLUSÃO: A extensão do regime de imunossupressão associado a baixas doses de irradiação diminui a gravidade da reação do enxerto versus hospedeiro, não abolida pelo uso de medula óssea depletada.

Haematopoietic stem cell transplantation in Switzerland. Report from the Swiss Transplant Working Group Blood and Marrow Transplantation (STABMT) Registry 1997-2003

In 1997, the Swiss Transplant Working Group Blood and Marrow Transplantation (STABMT) initiated a mandatory national registry for all haematopoietic stem cell transplants (HSCT) in Switzerland. As of 2003, information was collected of 2010 patients with a first HSCT (577 allogeneic (29%) and 1433 autologous (71%) HSCT) and 616 additional re-transplants. This included 1167 male and 843 female patients with a median age of 42.4 years (range 0.2-76.6 years). Main indications were leukaemias (592; 29%) lymphoproliferative disorders (1,061; 53%), solid tumours (295; 15%) and non-malignant disorders (62; 3%). At the time of analysis 1,263 patients were alive (63%), 747 had died (37%). Probability of survival, transplant related mortality or relapse at 5 years was 52%, 21%, 36% for allogeneic and 54%, 5%, 60% for autologous HSCT. Outcome depended on indication, donor type, stem cell source and age of patient. HSCT is an established therapy in Switzerland. These data describe current practice and outcome.

Optimization of a transplant model to assess skin reconstitution from stem cell-enriched primary human keratinocyte populations

Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated; and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.

Tissue and cell's trafficking: a byproduct of transplant activity's trash. The future is now. A Public Health problem

Transplantation is one of the most beautiful achievements for humanity in the last century and became the last hope to many patients. As other beautiful achievements, it has been used by criminals. The future of transplantation will be focused on tissue and cells transplantation. Trafficking of human beings to organ removal and trafficking of human organs are an early stage of trafficking on tissues and cells comparable with slaves trafficking in the 17th and 18th century. As 400 years ago, the motive for the crime is development, economy and profit. Transplant surgery is the modern “cotton gin” to this new commerce. Poverty exploitation, unprotected people, are always the victims. Even so, there are some differences since then. The paying buyers are the patients themselves and the “cotton” transplanted is not so harmless. Unsafe tissues and cells inappropriately collected and allocated can be so dangerous to the recipient and his family, that the dreamed transplant/implant becomes a nightmare. Beyond the trafficking crime, there is a most dangerous associated crime that is the crime of spreading dangerous infectious diseases.

Modulation of dermal microvascular endithelial cell responses to growth factors and haemostatic factors in the presence of vitronectin

In order to effect permanent closure in burns patients suffering from full thickness wounds, replacing their skin via split thickness autografting, is essential. Dermal substitutes in conjunction with widely meshed split thickness autografts (+/- cultured keratinocytes) reduce scarring at the donor and recipient sites of burns patients by reducing demand for autologous skin (both surface area and thickness), without compromising dermal delivery at the wound face. Tissue engineered products such as Integra consist of a dermal template which is rapidly remodelled to form a neodermis, at which time the temporary silicone outer layer is removed and replaced with autologous split thickness skin. Whilst provision of a thick tissue engineered dermis at full thickness burn sites reduces scarring, it is hampered by delays in vascularisation which results in clinical failure. The ultimate success of any skin graft product is dependent upon a number of basic factors including adherence, haemostasis and in the case of viable tissue grafts, success is ultimately dependent upon restoration of a normal blood supply, and hence this study. Ultimately, the goal of this research is to improve the therapeutic properties of tissue replacements, through impregnation with growth factors aimed at stimulating migration and proliferation of microvascular endothelial cells into the donor tissue post grafting. For the purpose of my masters, the aim was to evaluate the responsiveness of a dermal microvascular endothelial cell line to growth factors and haemostatic factors, in the presence of the glycoprotein vitronectin. Vitronectin formed the backbone for my hypothesis and research due to its association with both epithelial and, more specifically, endothelial migration and proliferation. Early work using a platform technology referred to as VitroGro (Tissue Therapies Ltd), which is comprised of vitronectin bound BP5/IGF-1, aided keratinocyte proliferation. I hypothesised that this result would translate to another epithelium - endothelium. VitroGro had no effect on endothelial proliferation or migration. Vitronectin increases the presence of Fibroblast Growth Factor (FGF) and Vascular Endothelial Growth Factor (VEGF) receptors, enhancing cell responsiveness to their respective ligands. So, although Human Microvascular Endothelial Cell line 1 (HMEC-1) VEGF receptor expression is generally low, it was hypothesised that exposure to vitronectin would up-regulate this receptor. HMEC-1 migration, but not proliferation, was enhanced by vitronectin bound VEGF, as well as vitronectin bound Epidermal Growth Factor (EGF), both of which could be used to stimulate microvascular endothelial cell migration for the purpose of transplantation. In addition to vitronectin's synergy with various growth factors, it has also been shown to play a role in haemostasis. Vitronectin binds thrombin-antithrombin III (TAT) to form a trimeric complex that takes on many of the attributes of vitronectin, such as heparin affinity, which results in its adherence to endothelium via heparan sulfate proteoglycans (HSP), followed by unaltered transcytosis through the endothelium, and ultimately its removal from the circulation. This has been documented as a mechanism designed to remove thrombin from the circulation. Equally, it could be argued that it is a mechanism for delivering vitronectin to the matrix. My results show that matrix-bound vitronectin dramatically alters the effect that conformationally altered antithrombin three (cATIII) has on proliferation of microvascular endothelial cells. cATIII stimulates HMEC-1 proliferation in the presence of matrix-bound vitronectin, as opposed to inhibiting proliferation in its absence. Binding vitronectin to tissues and organs prior to transplant, in the presence of cATIII, will have a profound effect on microvascular infiltration of the graft, by preventing occlusion of existing vessels whilst stimulating migration and proliferation of endothelium within the tissue.

A brief history of haemotopoietic stem cell Ex vivo expansion

Haematopoiesis is the process by which a hierarchy of mature and progenitor blood cells are formed. These cell populations are all derived from multipotent haematopoietic stem cells (HSC), which reside in the bone marrow ‘niche’ of adult humans. Over the lifetime of a healthy individual, this HSC population replenishes between 1010-1011 blood cells on a daily basis. Dysregulation of this system can lead to a number of haematopoietic diseases, including aplastic anaemias and leukaemias, which result in, or require for disease resolution, bone marrow cell depletion. In 1956, E. Donnall Thomas demonstrated that haematopoiesis could be restored by transplanting bone marrow-derived cells from one man into his identical twin brother, who was suffering from advanced leukaemia. His success drew significant interest in academic research and medicine communities, and 12 years later, the first successful allogeneic transplant was performed. To this day, HSCs remain the most studied and characterised stem cell population. In fact, HSCs are the only stem cell population routinely utilised in the clinic. As such, HSCs function as a model system both for the biological investigation of stem cells, as well as for their clinical application. Herein, we briefly review HSC transplantation, strategies for the ex vivo cultivation of HSCs, recent clinical outcomes, and their impact on the future direction of HSC transplantation therapy.

Development of an ultra-thin fibroin membrane for RPE cell transplantation

Purpose: One of the challenges associated with cell-based therapies for repairing the retina is the development of suitable materials on which to grow and transplant retinal cells. Using the ARPE-19 cell line, we have previously demonstrated the feasibility of growing RPE-derived cells on membranes prepared from the silk protein fibroin. The present study was aimed at developing a porous, ultra-thin fibroin membrane that might better support development of apical-basal polarity in culture, and to extend this work to primary cultures of human RPE cells. Methods: Ultra-thin fibroin membranes were prepared using a highly polished casting table coated with Topas® (a cyclic olefin copolymer) and a 1:0.03 aqueous solution of fibroin and PEO (Mv 900 000 g/mol). Following drying, the membranes were water annealed to make them water-stable, washed in water to remove PEO, sterilised by treatment with 95% ethanol, and washed extensively in saline. Primary cultures containing human RPE cells were established from donor posterior eye cups and maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum and antibiotics. First passage cultures were seeded onto fibroin membranes pre-coated with vitronectin and grown for 6 weeks in medium supplemented with 1% serum. Comparative cultures were established on porous 1.0 µm pore PET membrane (Millipore) and using ARPE-19 cells. Results: The fibroin membranes displayed an average thickness of 3 µm and contained numerous dimples/pore-like structures of up to 3-5 µm in diameter. The primary cultures predominantly contained pigmented epithelial cells, but mesenchymal cells (presumed fibroblasts) were also often present. Passaged cultures appeared to attach equally well to either fibroin or PET membranes. Over time cells on either material adopted a more cobblestoned morphology. Conclusions: Progress has been made towards developing a porous ultra-thin fibroin membrane that supports cultivation of RPE cells. Further studies are required to determine the degree of membrane permeability and RPE polarity.

Mesenchymal stromal cells transiently alter the inflammatory milieu post-transplant to delay graft-versus-host disease

Background: Multipotent mesenchymal stromal cells suppress T-cell function in vitro, a property that has underpinned their use in treating clinical steroid-refractory graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. However the potential of mesenchymal stromal cells to resolve graft-versus-host disease is confounded by a paucity of pre-clinical data delineating their immunomodulatory effects in vivo. Design and Methods: We examined the influence of timing and dose of donor-derived mesenchymal stromal cells on the kinetics of graft-versus-host disease in two murine models of graft-versus-host disease (major histocompatibility complex-mismatched: UBI-GFP/BL6 [H-2b]→BALB/c [H-2d] and the sibling transplant mimic, UBI-GFP/BL6 [H-2b]→BALB.B [H-2b]) using clinically relevant conditioning regimens. We also examined the effect of mesenchymal stromal cell infusion on bone marrow and spleen cellular composition and cytokine secretion in transplant recipients. Results: Despite T-cell suppression in vitro, mesenchymal stromal cells delayed but did not prevent graft-versus-host disease in the major histocompatibility complex-mismatched model. In the sibling transplant model, however, 30% of mesenchymal stromal cell-treated mice did not develop graft-versus-host disease. The timing of administration and dose of the mesenchymal stromal cells influenced their effectiveness in attenuating graft-versus-host disease, such that a low dose of mesenchymal stromal cells administered early was more effective than a high dose of mesenchymal stromal cells given late. Compared to control-treated mice, mesenchymal stromal cell-treated mice had significant reductions in serum and splenic interferon-γ, an important mediator of graft-versus-host disease. Conclusions: Mesenchymal stromal cells appear to delay death from graft-versus-host disease by transiently altering the inflammatory milieu and reducing levels of interferon-γ. Our data suggest that both the timing of infusion and the dose of mesenchymal stromal cells likely influence these cells’ effectiveness in attenuating graft-versus-host disease.

Modern diagnosis of Epstein-Barr virus infections and post-transplant lymphoproliferative disease

Infection by Epstein-Barr virus (EBV) occurs in approximately 95% of the world s population. EBV was the first human virus implicated in oncogenesis. Characteristic for EBV primary infection are detectable IgM and IgG antibodies against viral capsid antigen (VCA). During convalescence the VCA IgM disappears while the VCA IgG persists for life. Reactivations of EBV occur both among immunocompromised and immunocompetent individuals. In serological diagnosis, measurement of avidity of VCA IgG separates primary from secondary infections. However, in serodiagnosis of mononucleosis it is quite common to encounter, paradoxically, VCA IgM together with high-avidity VCA IgG, indicating past immunity. We determined the etiology of this phenomenon and found that, among patients with cytomegalovirus (CMV) primary infection a large proportion (23%) showed antibody profiles of EBV reactivation. In contrast, EBV primary infection did not appear to induce immunoreactivation of CMV. EBV-associated post-transplant lymphoproliferative disease (PTLD) is a life threatening complication of allogeneic stem cell or solid organ transplantation. PTLD may present with a diverse spectrum of clinical symptoms and signs. Due to rapidity of PTLD progression especially after stem cell transplantation, the diagnosis must be obtained quickly. Pending timely detection, the evolution of the fatal disease may be halted by reduction of immunosuppression. A promising new PTLD treatment (also in Finland) is based on anti-CD-20 monoclonal antibodies. Diagnosis of PTLD has been demanding because of immunosuppression, blood transfusions and the latent nature of the virus. We set up in 1999 to our knowledge first in Finland for any microbial pathogen a real-time quantitative PCR (qPCR) for detection of EBV DNA in blood serum/plasma. In addition, we set up an in situ hybridisation assay for EBV RNA in tissue sections. In collaboration with a group of haematologists at Helsinki University Central Hospital we retrospectively determined the incidence of PTLD among 257 allogenic stem cell transplantations (SCT) performed during 1994-1999. Post-mortem analysis revealed 18 cases of PTLD. From a subset of PTLD cases (12/18) and a series of corresponding controls (36), consecutive samples of serum were studied by the new EBV-qPCR. All the PTLD patients were positive for EBV-DNA with progressively rising copy numbers. In most PTLD patients EBV DNA became detectable within 70 days of SCT. Of note, the appearance of EBV DNA preceded the PTLD symptoms (fever, lymphadenopathy, atypical lymphocytes). Among the SCT controls, EBV DNA occurred only sporadically, and the EBV-DNA levels remained relatively low. We concluded that EBV qPCR is a highly sensitive (100%) and specific (96%) new diagnostic approach. We also looked for and found risk factors for the development of PTLD. Together with a liver transplantation group at the Transplantation and Liver Surgery Clinic we wanted to clarify how often and how severely do EBV infections occur after liver transplantation. We studied by the EBV qPCR 1284 plasma samples obtained from 105 adult liver transplant recipients. EBV DNA was detected in 14 patients (13%) during the first 12 months. The peak viral loads of 13 asymptomatic patients were relatively low (<6600/ml), and EBV DNA subsided quickly from circulation. Fatal PTLD was diagnosed in one patient. Finally, we wanted to determine the number and clinical significance of EBV infections of various types occurring among a large, retrospective, nonselected cohort of allogenic SCT recipients. We analysed by EBV qPCR 5479 serum samples of 406 SCT recipients obtained during 1988-1999. EBV DNA was seen in 57 (14%) patients, of whom 22 (5%) showed progressively rising and ultimately high levels of EBV DNA (median 54 million /ml). Among the SCT survivors, EBV DNA was transiently detectable in 19 (5%) asymptomatic patients. Thereby, low-level EBV-DNA positivity in serum occurs relatively often after SCT and may subside without specific treatment. However, high molecular copy numbers (>50 000) are diagnostic for life-threatening EBV infection. We furthermore developed a mathematical algorithm for the prediction of development of life-threatening EBV infection.

Detection of recently discovered human polyomaviruses in a longitudinal kidney transplant cohort

A large number of human polyomaviruses have been discovered in the last 7 years. However, little is known about the clinical impact on vulnerable immunosuppressed patient populations. Blood, urine, and respiratory swabs collected from a prospective, longitudinal adult kidney transplant cohort (n = 167) generally pre-operatively, at day 4, months 1, 3, and 6 posttransplant, and at BK viremic episodes within the first year were screened for 12 human polyomaviruses using real-time polymerase chain reaction. Newly discovered polyomaviruses were most commonly detected in the respiratory tract, with persistent shedding seen for up to 6 months posttransplant. Merkel cell polyomavirus was the most common detection, but was not associated with clinical symptoms or subsequent development of skin cancer or other skin abnormalities. In contrast, KI polyomavirus was associated with respiratory disease in a subset of patients. Human polyomavirus 9, Malawi polyomavirus, and human polyomavirus 12 were not detected in any patient samples.