Towards the development of a bioengineered uterus: comparison of different protocols for rat uterus decellularization.

Uterus transplantation (UTx) may be the only possible curative treatment for absolute uterine factor infertility, which affects 1 in every 500 females of fertile age. We recently presented the 6-month results from the first clinical UTx trial, describing nine live-donor procedures. This routine involves complicated surgery and requires potentially harmful immune suppression to prevent rejection. However, tissue engineering applications using biomaterials and stem cells may replace the need for a live donor, and could prevent the required immunosuppressive treatment. To investigate the basic aspects of this, we developed a novel whole-uterus scaffold design for uterus tissue engineering experiments in the rat. Decellularization was achieved by perfusion of detergents and ionic solutions. The remaining matrix and its biochemical and mechanical properties were quantitatively compared from using three different protocols. The constructs were further compared with native uterus tissue composition. Perfusion with Triton X-100/dimethyl sulfoxide/H2O led to a compact, weaker scaffold that showed evidence of a compromised matrix organization. Sodium deoxycholate/dH2O perfusion gave rise to a porous scaffold that structurally and mechanically resembled native uterus better. An innovative combination of two proteomic analyses revealed higher fibronectin and versican content in these porous scaffolds, which may explain the improved scaffold organization. Together with other important protocol-dependent differences, our results can contribute to the development of improved decellularization protocols for assorted organs. Furthermore, our study shows the first available data on decellularized whole uterus, and creates new opportunities for numerous in vitro and in vivo whole-uterus tissue engineering applications.

Human liver sinusoidal endothelial cells induce apoptosis in activated T cells: a role in tolerance induction.

BACKGROUND: The liver may have a role in peripheral tolerance, by serving as a site for trapping, apoptosis and phagocytosis of activated T cells. It is not known which hepatic cells are involved in these processes. It was hypothesised that liver sinusoidal endothelial cells (LSEC) which are strategically placed for participation in the regulation of sinusoidal blood flow, and express markers involved in recognition, sequestration and apoptosis, may contribute to peripheral tolerance by inducing apoptosis of activated T cells. METHODS: By using immunoassays and western blot analysis, the fate of activated T cells when incubated with human LSEC isolated from normal healthy livers was investigated. RESULTS: Evidence that activated (approximately 30%) but not non-activated T cells undergo apoptosis on incubation with human LSEC in mixed cell cultures is provided. No difference in the results was observed when unstimulated and cytokine-stimulated LSEC were used. T cell-LSEC contact is required for induction of apoptosis. Apoptosis induced by LSEC was associated with caspase 8 and 3 activity and strong expression of the proapoptotic molecule Bak. Transforming growth factor beta (TGFbeta) produced constitutively by LSEC is partly responsible for the caspase-induced apoptosis, as neutralising antibodies to TGFbeta markedly attenuated apoptosis, up regulated the antiapoptotic molecule Bcl-2 and partially blocked caspase-3 activity. CONCLUSION: These findings broaden the potential role of LSEC in immune tolerance and homeostasis of the immune system. This study may provide insight for exploring the mechanisms of immune tolerance by liver allografts, immune escape by some liver pathogens including hepatitis C and pathogenesis of liver diseases.

Antibodies to liver sinusoidal endothelial cells modulate immune responses in liver transplantation.

AIM: Liver sinusoidal endothelial cells (LSECs) have been implicated to play a role in the induction of liver allograft rejections. Here, we studied the clinical consequences of preformed LSEC-reactive antibodies and their functional capacity in modulating T-cell responses. METHODS: Pre- and posttransplant sera and T lymphocytes from 95 liver transplant patients were used in this study. LSECs were isolated from a normal healthy liver. Binding of antibodies to LSECs was detected using flow cytometric analysis. To study whether LSEC antibodies facilitated cell-mediated immunity, a mixed cell culture (MCC) assay was used. Cytokines in the supernatant of MCC were also measured by enzyme-linked immunosorbent assay. Immunohistochemical staining on liver biopsy sections was performed to detect deposition of immunoglobulins in LSEC during rejections. RESULTS: Significantly higher numbers of patients with rejections had LSEC antibodies (35/50, 70%) compared with 8/45 (18%) without rejections (P < .0001). Purified fractions of LSEC antibodies induced the expression of the costimulatory marker CD86 on LSECs. Significantly higher numbers of patients with LSEC antibodies and rejections had an increased proliferation of T cells and markedly decreased levels of transforming growth factor (TGF)-beta in the MCC as compared with those without antibodies and rejections (P < .0001, P < .0001, respectively). Deposition of antibodies in LSECs during rejection episodes was observed in the biopsies of patients with LSEC antibodies but not in those without LSEC antibodies. CONCLUSION: Our data suggest that antibodies to LSEC may facilitate acute liver allograft rejections by down-regulating the immune modulating cytokine TGF-beta and thus up-regulating alloreactive T-cell proliferation.

Identification of expandable human hepatic progenitors which differentiate into mature hepatic cells in vivo.

BACKGROUND: Liver diseases include a wide spectrum of both acute and chronic conditions which are associated with significant morbidity and mortality worldwide. Hepatocyte transplantation has therapeutic potential in the treatment of liver diseases, but its clinical use is hampered by the lack of donor tissue. Generation of hepatocytes in vitro from adult or fetal liver cell progenitors or, alternatively, identification of a progenitor population which in vivo can generate mature liver cells could solve this problem. METHODS: CD117+/CD34+/Lin- human fetal liver cells were isolated by magnetic cell sorting and expanded in culture. Both freshly isolated and in vitro expanded cells in various passages were studied for their ability to be functional in hepatic parenchyma following d-galactosamine (GalN) induced injury in nude C57 black mice. RESULTS: Freshly isolated and in vitro expanded CD117+/CD34+/Lin- cells, when transplanted intrasplenically into GalN treated mice, morphologically and functionally differentiated into hepatocytes and cholangiocytes. Human specific albumin, alpha fetoprotein, cytokeratin 19, and antitrypsin mRNA were expressed in mouse liver. In addition, the human progenitor cells expressed glucose-6-phosphatase, glycogen, albumin, gamma glutamyl transpeptidase, and dipeptidyl peptidase IV after transplantation. Expanded cells in various passages maintained their capacity to differentiate into functional liver cells. CONCLUSIONS: Fetal liver CD117+/CD34+/Lin- progenitors and their progeny proliferated in vitro and also functionally differentiated into mature hepatic cells in an acute liver injury model. Successful in vitro expansion of liver progenitor cells provides a basis for developing cell therapy strategies, metabolic and toxicity testing systems, and may serve as a vehicle for gene therapy.

Identification of the nonclassical HLA molecules, mica, as targets for humoral immunity associated with irreversible rejection of kidney allografts.

BACKGROUND: A substantial portion of kidney allografted patients experience early acute rejection episodes and even irreversible rejections in the early posttransplantation period. The presence of HLA alloantibodies before grafting is associated with early immunological complications, but in many patients rejections and graft loss occur even in the absence of such antibodies. METHODS: In this study, 748 serum samples taken before and at various time points after kidney transplantation from 139 patients were investigated for the presence, frequency, and specificity of kidney microvascular endothelial cell (KMEC)-reactive antibodies using major histocompatability class (MHC) I-related chain A (MICA) transfected cells and flow cytometry, antibody blocking experiments, and Western blotting. The ability of MICA-specific antibodies to fix complement and to induce a prothrombotic phenotype in KMECs was investigated. RESULTS: A polymorphic, 62 kDa nonclassical HLA class I molecule is identified as a new target molecule for reactivity in sera from patients with irreversible rejections. Specific blocking and transfection experiments verified the target molecule as MICA. A significant correlation was established for pre- or posttransplantation MICA humoral immunity and graft loss (P<0.001). MICA-specific antibody titers increased in the posttransplantation period and were present before any signs of clinical rejection. MICA antibody-containing patient sera induced a prothrombotic phenotype in KMECs. CONCLUSION: The increasing polymorphism detected at the MIC loci combined with the results of this study suggest that typing for the MIC loci and crossmatching for the detection of anti-MIC antibodies before transplantation should be used routinely. A better recipient-donor selection based on a negative crossmatch for both anti-donor HLA and MICA antibodies will decrease early graft rejections and losses.

High frequency of autoantibodies in patients with primary sclerosing cholangitis that bind biliary epithelial cells and induce expression of CD44 and production of interleukin 6.

AIM: Sera of patients with autoimmune liver diseases were investigated for the presence of autoantibodies binding to human biliary epithelial cells (BECs). Furthermore, their functional capacity was investigated by testing their capacity to fix complement as well as induce expression of various adhesion molecules and production of cytokines. METHODS: Sera from patients with various stages of primary sclerosing cholangitis (PSC; n=30), primary biliary cirrhosis (PBC; n=29), autoimmune hepatitis (AIH; n=25), and normal controls (n=12) were investigated for the presence of antibodies that reacted with unstimulated and cytokine stimulated BECs isolated from a normal healthy liver. To demonstrate organ specificity, lung epithelial cells (LECs) were used as control cells. Antibodies were tested for their functional capacity. RESULTS: Compared with controls (8%), significantly higher numbers of PSC patients (63%, p=0.001), but not PBC (37%, NS) or AIH (16%, NS) patients, had anti-BEC antibodies. In 90% of PSC patients, the autoantibodies reacted only with cytokine stimulated target cells. Lower numbers of PSC (6%), PBC (10%), and AIH (0%) patients had LEC antibodies. Other significant findings were that anti-BEC antibodies were found in (i) PSC patients with either the HLA-DRB1*0301 or DR2 allele compared with those without (p=0.007); and (ii) in PBC patients with end stage disease compared with those without (p=0.018). Furthermore, anti-BEC antibodies from PSC and PBC but not AIH patients induced BECs to produce high levels of the cytokine interleukin 6. IgM and IgG fractions isolated from PSC but not PBC and AIH sera induced significantly increased expression of the cell adhesion molecule CD44. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blot analysis of BEC membranes demonstrated a specific band of 40 kDa with PSC sera and 45, 42, 30, and 33 kDa bands with PBC sera, which were absent in control groups. CONCLUSION: Thus for the first time we have demonstrated the presence of functionally important autoantibodies to cell surface expressed antigens on the relevant target cells of destruction, namely BECs, in PSC and PBC. These finding have important implications for the pathogenesis of bile duct destruction in these patients.

The in vitro activity and specificity of human endothelial cell-specific promoters in porcine cells.

The chronic shortage of human organs, tissues and cells for transplantation has inspired research on the possibility of using animal donor tissue instead. Transplantation over a species barrier is associated with rejections which are difficult to control. Therefore, it is generally agreed that successful pig to human xenotransplantation requires donor pigs to be genetically modified. Vascular endothelium is the most immediate barrier between the xenogeneic donor organ and host immune and nonimmune defense systems. Thus, these cells are the prime targets for such genetic modifications. Luciferase assays were used to evaluate the activity and specificity of human endothelial-cell specific promoters in porcine aortic-, microvascular- and nonendothelial cells. The promoters for human Flk-1 (fetal liver kinase-1), Flt-1 (fms-like tyrosine kinase), ICAM-2 (intercellular adhesion molecule-2), thrombomodulin and vWf (von Willebrand factor) supported similar levels of luciferase expression in human and porcine aortic endothelial cells, with the Flk-1 promoter being the strongest followed by the thrombomodulin promoter. Relative to the activity of the CMV promoter, the human endothelial cell-specific promoters all showed less activity in porcine kidney microvascular endothelial cells than in liver or brain microvascular endothelial cells. The thrombomodulin and Flk-1 promoters exhibited similar activity in liver and kidney microvascular endothelial cells, whereas the Flk-1 promoter was stronger in aortic and brain microvascular endothelial cells. Human endothelial cell-specific promoters also showed some degree of specificity in pig, because they supported less luciferase activity in porcine nonendothelial cell lines. Based on the in vitro data and previously published in vivo data, the human Flk-1 and thrombomodulin promoters are good candidate promoters for strong endothelial cell-specific gene expression in transgenic pigs.

Tumour necrosis factor alpha impairs function of liver derived T lymphocytes and natural killer cells in patients with primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is considered to be a chronic autoimmune disease where infiltrating T lymphocytes have been implicated in the destruction of bile ducts. Altered function of these T cells may reflect abnormalities in the immune response leading to tissue damage. AIM: We investigated the proliferative and functional capacity of freshly isolated liver derived T lymphocytes (LDLs) and natural killer (NK) cells from PSC patients. METHODS: The proliferative responses to common mitogens such as phytohaemagglutinin (PHA), concanavalin A (Con A), and lipopolysaccharide (LPS) were studied, and the cytotoxic function of T lymphocytes was measured using allogeneic target cells. NK (CD56(+)/16(+)) cytotoxic function was measured using the two cell lines K562 (NK sensitive) and Raji lymphoma cells (NK resistant). RESULTS: Compared with patients with primary biliary cirrhosis (PBC), autoimmune hepatitis (AIH), and normal controls (without liver disease), in PSC: (1) LDLs contained a low percentage of T cells; (2) there was significantly decreased expression of interleukin (IL)-2 receptor (p<0.001) on activated T cells (HLA-DR(+)); (3) LDLs but not peripheral blood lymphocytes had significantly impaired proliferative responses to mitogens such as PHA, Con A, and LPS (p< 0.001); (4) no cytotoxic activity of PSC liver T and NK cells was recorded; (5) significantly higher levels of tumour necrosis factor alpha (TNF-alpha) and IL-1beta but lower levels of IL-2, IL-10, and interferon gamma were found in the supernatants of mitogen stimulated LDL cultures (p<0.001); (6) higher percentages of freshly isolated PSC LDLs contained intracytoplasmic TNF-alpha and IL-1beta; and (7) pretreatment of PSC LDLs in vitro with neutralising TNF antibodies significantly enhanced proliferative responses and allowed IL-2 receptor expression following stimulation. In addition, the impaired cytolytic activity of both NK and T cells was partially restored. Impaired proliferative or functional capacity of liver derived T cells was not observed in either PBC or AIH patients. CONCLUSIONS: We suggest that reduced T cell reactivity in liver infiltrating cells obtained from patients with PSC is due to high local production of TNF-alpha. Our findings indicate that the use of anti-TNF antibodies as an alternative treatment for PSC patients should be evaluated.

HLA-specific alloantibodies and renal graft outcome.

HLA-specific humoral immunity, as a result of recipient allosensitization, induces hyperacute rejection of allogenic kidney grafts. Cross-match tests are performed to avoid this complication. However, current techniques do not allow determination of HLA-specificity of donor-reactive antibodies in the acute cadaver-donor situation. New methods are described and discussed in this report as well as the alloantibody specificities that are of clinical importance. Alloantibodies not only mediate hyperacute rejection but may also participate in the acute rejection of organ grafts. Clinical associations between early immunological complications, such as acute rejection, in heart, liver and kidney allografted patients and pre-transplantation humoral alloimmunity emphasize the need for proper determination of donor-specific humoral immunity prior to transplantation.