In vitro development of islets from human adult pancreatic tissues

Transplantation of isolated islets from cadaver pancreas is a promising possibility for the optimal treatment of type 1 diabetes. The lack of islets is a major problem. Here we have investigated the possibility of generating islets in tissue culture of human pancreatic cells. We first reproduced a previously reported method of in vitro generation of endocrine cells from human adult pancreatic tissue. By tracing the bromodeoxyuridine-labeled cells in differentiated islet buds, we found that the pancreatic progenitor cells represented a subpopulation of cytokeratin 19 (CK19)-positive ductal cells. Serum-free medium and Matrigel overlay were essential for the endocrine differentiation. We then examined the involvement of preexisting islet cells in islet neogenesis. About 6-10% of endocrine cells dedifferentiated and acquired a transitional phenotype by coexpressing CK19. Significant cell proliferation was only observed in CK19-positive cells, but not in chromogranin A-positive endocrine cells. The in vitro-derived human islets were morphologically and functionally immature when compared with normal islets. Their insulin mRNA levels were only 4-5% of that found in fresh human islets, and glucose-stimulated insulin release was 3 times lower than that of control islets. Moreover, some immature endocrine cells coexpressed insulin and glucagon. After transplantation in nude mice, the in vitro-generated islets became mature with one type of hormone per endocrine cell. In addition, we also found that also in both fresh islet transplants many cells coexpressed endocrine markers and ductal marker CK19 as a sign of ductal to endocrine cell transition. Finally, we studied the effects of clinically used immunosuppressive drugs on precursor cell proliferation and differentiation. Mycophenolate mofetil (MMF) severely hampered duct-cell proliferation, and significantly reduced the total DNA content indicating its antiproliferative effect on the precursors. Tacrolimus mainly affected differentiated beta cells by decreasing the insulin content per DNA as well as the proportion of insulin-positive cells. Sirolimus and daclizumab did not show any individual or synergistic side effects suggesting that these drugs are amenable for use in clinical islet transplantation. In summary, we confirm the capacity of endocrine differentiation from progenitors present in the adult human pancreas. The plasticity of differentiated cell types of human pancreas may be a potential mechanism of human pancreas regeneration. Ductal cell differentiation into endocrine cells in transplanted islets may be an important factor in sustaining the long-term function of islet transplants. The immunosuppressive protocol is likely to be an important determinant of long-term clinical islet graft function. Moreover, these results provide new information on the mechanisms of pancreatic islet regeneration and provide the basis for the development of new strategies for the treatment of insulin deficient diabetes mellitus.

VEGFR-3 and Tie pathways in vascular network formation

The blood and lymphatic vascular systems are essential for life, but they may become harnessed for sinister purposes in pathological conditions. For example, tumors learn to grow a network of blood vessels (angiogenesis), securing a source of oxygen and nutrients for sustained growth. On the other hand, damage to the lymph nodes and the collecting lymphatic vessels may lead to lymphedema, a debilitating condition characterized by peripheral edema and susceptibility to infections. Promoting the growth of new lymphatic vessels (lymphangiogenesis) is an attractive approach to treat lymphedema patients. Angiopoietin-1 (Ang1), a ligand for the endothelial receptor tyrosine kinases Tie1 and Tie2. The Ang1/Tie2 pathway has previously been implicated in promoting endothelial stability and integrity of EC monolayers. The studies presented here elucidate a novel function for Ang1 as a lymphangiogenic factor. Ang1 is known to decrease the permeability of blood vessels, and could thus act as a more global antagonist of plasma leakage and tissue edema by promoting growth of lymphatic vessels and thereby facilitating removal of excess fluid and other plasma components from the interstitium. These findings reinforce the idea that Ang1 may have therapeutic value in conditions of tissue edema. VEGFR-3 is present on all endothelia during development, but in the adult its expression becomes restricted to the lymphatic endothelium. VEGF-C and VEGF-D are ligands for VEGFR-3, and potently promote lymphangiogenesis in adult tissues, with direct and remarkably specific effects on the lymphatic endothelium in adult tissues. The data presented here show that VEGF-C and VEGF-D therapy can restore collecting lymphatic vessels in a novel orthotopic model of breast cancer-related lymphedema. Furthermore, the study introduces a novel approach to improve VEGF-C/VEGF-D therapy by using engineered heparin-binding forms of VEGF-C, which induced the rapid formation of organized lymphatic vessels. Importantly, VEGF-C therapy also greatly improved the survival and integration of lymph node transplants. The combination of lymph node transplantation and VEGF-C therapy provides a basis for future therapy of lymphedema. In adults, VEGFR-3 expression is restricted to the lymphatic endothelium and the fenestrated endothelia of certain endocrine organs. These results show that VEGFR-3 is induced at the onset of angiogenesis in the tip cells that lead the formation of new vessel sprouts, providing a tumor-specific vascular target. VEGFR-3 acts downstream of VEGF/VEGFR-2 signals, but, once induced, can sustain angiogenesis when VEGFR-2 signaling is inhibited. The data presented here implicate VEGFR-3 as a novel regulator of sprouting angiogenesis along with its role in regulating lymphatic vessel growth. Targeting VEGFR-3 may provide added efficacy to currently available anti-angiogenic therapeutics, which typically target the VEGF/VEGFR-2 pathway.

New molecular strategies for prevention of fibroproliferative vascular disease : An experimental approach to restenosis

Vascular intimal hyperplasia is a major complication following angioplasty. The hallmark feature of this disorder is accumulation of dedifferentiated smooth muscle cells (SMCs) to the luminal side of the injured artery, cellular proliferation, migration, and synthesis of extracellular matrix. This finally results in intimal hyperplasia, which is currently considered an untreatable condition. According to current knowledge, a major part of neointimal cells derive from circulating precursor cells. This has outdated the traditional in vitro cell culture methods of studying neointimal cell migration and proliferation using cultured medial SMCs. Somatostatin and some of its analogs with different selectivity for the five somatostatin receptors (sst1 through sst5) have been shown to have vasculoprotective properties in animal studies. However, clinical trials using analogs selective for sst2/sst3/sst5 to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) have failed to show any major benefits. Sirolimus is a cell cycle inhibitor that has been suggested to act synergistically with the protein-tyrosine kinase inhibitor imatinib to inhibit intimal hyperplasia in rat already at well-tolerated submaximal oral doses. The mechanisms behind this synergy and its long-term efficacy are not known. The aim of this study was to set up an ex vivo vascular explant culture model to measure neointimal cell activity without excluding the participation of circulating progenitor cells. Furthermore, two novel potential vasculoprotective treatment strategies were evaluated in detail in rat models of intimal hyperplasia and in the ex vivo explant model: sst1/sst4-selective somatostatin receptor analogs and combination treatment with sirolimus and imatinib. This study shows how whole vessel explants can be used to study the kinetics of neointimal cells and their progenitors, and to evaluate the anti-migratory and anti-proliferative properties of potential vasculoprotective compounds. It also shows how the influx of neointimal progenitor cells occurs already during the first days after vascular injury, how the contribution of cell migration is more important in the injury response than cell proliferation, and how the adventitia actively contribute in vascular repair. The vasculoprotective effect of somatostatin is mediated preferentially through sst4, and through inhibition of cell migration rather than of proliferation, which may explain why sst2/sst3/sst5-selective analogs have failed in clinical trials. Furthermore, a brief early oral treatment with the combination of sirolimus and imatinib at submaximal doses results in long-term synergistic suppression of intimal hyperplasia. The synergy is a result of inhibition of post-operative thrombocytosis and leukocytosis, inhibition of neointimal cell migration to the injury-site, and maintenance of cell integrity by inhibition of apoptosis and SMC dedifferentiation. In conclusion, the influx of progenitor cells already during the first days after injury and the high neointimal cell migratory activity underlines the importance of early therapeutic intervention with anti-migratory compounds to prevent neointimal hyperplasia. Sst4-selective analogs and the combination therapy with sirolimus and imatinib represent potential targets for the development of such vasculoprotective therapies.

Genetically Engineered Oncolytic Adenoviruses for the Treatment of Kidney and Breast Cancer

Metastatic kidney and breast cancer are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment are oncolytic adenoviruses, which have demonstrated excellent safety in many clinical trials. However, antitumor efficacy needs to be improved in order to make oncolytic viruses a viable treatment alternative. To be able to follow oncolytic virus replication in vivo, we set up a non-invasive imaging system based on coinjection of a replication deficient luciferase expressing virus and a replication competent virus. The system was validated in vitro and in vivo and used in other projects of the thesis. In another study we showed that capsid modifications on adenoviruses result in enhanced gene transfer and increased oncolytic effect on renal cancer cells in vitro. Moreover, capsid modified oncolytic adenoviruses demonstrated significantly improved antitumor efficacy in murine kidney cancer models. To transcriptionally target kidney cancer tissue we evaluated two hypoxia response elements for their usability as tissue specific promoters using a novel dual luciferase imaging system. Based on the results of the promoter evaluation and the studies on capsid modifications, we constructed a transcriptionally and transductionally targeted oncolytic adenovirus armed with an antiangiogenic transgene for enhanced renal cell cancer specificity and improved antitumor efficacy. This virus exhibited kidney cancer specific replication and significantly improved antitumor effect in a murine model of intraperitoneal disseminated renal cell cancer. Cancer stem cells are thought to be resistant to conventional cancer drugs and might play an important role in breast cancer relapse and the formation of metastasis. Therefore, we examined if capsid modified oncolytic adenoviruses are able to kill these cells proposed to be breast cancer initiating. Efficient oncolytic effect and significant antitumor efficacy on tumors established with breast cancer initiating cells was observed, suggesting that oncolytic adenoviruses might be able to prevent breast cancer relapse and could be used in the treatment of metastatic disease. In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of metastatic kidney and breast cancer.

Novel prognostic factors in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a malignant clonal blood disease that originates from a pluripotent hematopoietic stem cell. The cytogenetic hallmark of CML, the Philadelphia chromosome (Ph), is formed as a result of reciprocal translocation between chromosomes 9 and 22, which leads to a formation of a chimeric BCR-ABL fusion gene. The BCR-ABL protein is a constitutively active tyrosine kinase that changes the adhesion properties of cells, constitutively activates mitogenic signaling, enhances cell proliferation and reduces apoptosis. This results in leukemic growth and the clinical disease, CML. With the advent of targeted therapies against the BCR-ABL fusion protein, the treatment of CML has changed considerably during the recent decade. In this thesis, the clinical significance of different diagnostic methods and new prognostic factors in CML have been assessed. First, the association between two different methods for measuring CML disease burden (the RQ-PCR and the high mitotic index metaphase FISH) was assessed in bone marrow and peripheral blood samples. The correlation between positive RQ-PCR and metaphase FISH samples was high. However, RQ-PCR was more sensitive and yielded measurable transcripts in 40% of the samples that were negative by metaphase FISH. The study established a laboratory-specific conversion factor for setting up the International Scale when standardizing RQ-PCR measurements. Secondly, the amount of minimal residual disease (MRD) after allogeneic hematopoietic stem cell transplantation (alloHSCT) was determined. For this, metaphase FISH was done for the bone marrow samples of 102 CML patients. Most (68%), had no residual cells during the entire follow-up time. Some (12 %) patients had minor (<1%) MRD which decreased even further with time, whereas 19% had a progressive rise in MRD that exceeded 1% or had more than 1% residual cells when first detected. Residual cells did not become eradicated spontaneously if the frequency of Ph+ cells exceeded 1% during follow-up. Next, the impact of deletions in the derivative chromosome 9, was examined. Deletions were observed in 15% of the CML patients who later received alloHSCT. After alloHSCT, there was no difference in the total relapse rate in patients with or without deletions. Nor did the estimates of overall survival, transplant-related mortality, leukemia-free survival and relapse-free time show any difference between these groups. When conventional treatment regimens are used, the der(9) status could be an important criterion, in conjunction with other prognostic factors, when allogeneic transplantation is considered. The significance of der(9) deletions for patients treated with tyrosine kinase inhibitors is not clear and requires further investigation. In addition to the der(9) status of the patient, the significance of bone marrow lymphocytosis as a prognostic factor in CML was assessed. Bone marrow lymphocytosis during imatinib therapy was a positive predictive factor and heralded optimal response. When combined with major cytogenetic response at three months of treatment, bone marrow lymphocytosis predicted a prognostically important major molecular response at 18 months of imatinib treatment. Although the validation of these findings is warranted, the determination of the bone marrow lymphocyte count could be included in the evaluation of early response to imatinib treatment already now. Finally, BCR-ABL kinase domain mutations were studied in CML patients resistant against imatinib treatment. Point mutations detected in the kinase domain were the same as previously reported, but other sequence variants, e.g. deletions or exon splicing, were also found. The clinical significance of the other variations remains to be determined.

Vascular growth factors and progenitor cells in cardiac allograft arteriosclerosis

Heart transplantation is the only therapeutic modality for many end-stage heart diseases but poor long-term survival remains a challenging problem. This is mainly due to the development of cardiac allograft arteriosclerosis (TxCAD) that is an accelerated form of coronary artery disease. Both traditional cardiovascular and transplantation-related risk factors for TxCAD have been identified but options for therapy are limited. TxCAD involves dysfunction of cardiac allograft vascular cells. Activated endothelial cells (EC) regulate allograft inflammation and secrete smooth muscle cell (SMC) growth factors. In turn, SMC and their progenitors invade the intima of the injured vessels and occlude the affected coronary arteries. Different vascular growth factors have to be delicately regulated in normal vascular development. In the present study, experimental heterotopic transplantation models were used to study the role of angiogenic and pro-inflammatory vascular endothelial growth factor (VEGF), EC growth factor angiopoietin (Ang), and SMC mitogen platelet-derived growth factor (PDGF) in the development of TxCAD. Pharmacological and gene transfer approaches were used to target these growth factors and to assess their therapeutic potential. This study shows that alloimmune response in heart transplants upregulates VEGF expression, and induces allograft angiogenesis that involves donor-derived primitive EC. Intracoronary adenoviral VEGF gene transfer increased macrophage infiltration, intimal angiogenesis and TxCAD. VEGF inhibition with PTK787 decreased allograft inflammation and TxCAD, and simultaneous PDGF inhibition with imatinib further decreased TxCAD. Specific inhibition of two VEGF-receptors (VEGFR) decreased allograft inflammation and TxCAD, and VEGFR-2 inhibition normalized the density of primitive and mature capillaries in the allografts. Adenovirus-mediated transient Ang1 expression in the allograft had anti-inflammatory and anti-arteriosclerotic effects. Adeno-associated virus (AAV)-mediated prolonged Ang1 or Ang2 expression had similar anti-inflammatory effects. However, AAV-Ang1 activated allograft SMC whereas AAV-Ang2 had no effects on SMC activation and decreased the development of TxCAD. These studies indicate an interplay of inflammation, angiogenesis and arteriosclerosis in cardiac allografts, and show that vascular growth factors are important regulators in the process. Also, VEGF inhibition, PDGF inhibition and angiopoietin therapy with clinically-relevant pharmacological agents or novel gene therapy approaches may counteract vascular dysfunction in cardiac allografts, and have beneficial effects on the survival of heart transplant patients in the future.

A four-variable model for the pattern-forming mechanism in Hydra

A generalized Gierer-Meinhardt model has been used to account for the transplantation experiments in Hydra. In this model, a cross inhibition between the two organizing centres (namely, head and foot) are assumed to be the only mode of interaction in setting up a stable morphogen distribution for the pattern formation in Hydra.

Dasatinib, nilotinib and standard-dose imatinib for the first line treatment of chronic myeloid leukaemia: Systematic reviews and economic analyses

- Background Nilotinib and dasatinib are now being considered as alternative treatments to imatinib as a first-line treatment of chronic myeloid leukaemia (CML). - Objective This technology assessment reviews the available evidence for the clinical effectiveness and cost-effectiveness of dasatinib, nilotinib and standard-dose imatinib for the first-line treatment of Philadelphia chromosome-positive CML. - Data sources Databases [including MEDLINE (Ovid), EMBASE, Current Controlled Trials, ClinicalTrials.gov, the US Food and Drug Administration website and the European Medicines Agency website] were searched from search end date of the last technology appraisal report on this topic in October 2002 to September 2011. - Review methods A systematic review of clinical effectiveness and cost-effectiveness studies; a review of surrogate relationships with survival; a review and critique of manufacturer submissions; and a model-based economic analysis. - Results Two clinical trials (dasatinib vs imatinib and nilotinib vs imatinib) were included in the effectiveness review. Survival was not significantly different for dasatinib or nilotinib compared with imatinib with the 24-month follow-up data available. The rates of complete cytogenetic response (CCyR) and major molecular response (MMR) were higher for patients receiving dasatinib than for those with imatinib for 12 months' follow-up (CCyR 83% vs 72%, p < 0.001; MMR 46% vs 28%, p < 0.0001). The rates of CCyR and MMR were higher for patients receiving nilotinib than for those receiving imatinib for 12 months' follow-up (CCyR 80% vs 65%, p < 0.001; MMR 44% vs 22%, p < 0.0001). An indirect comparison analysis showed no difference between dasatinib and nilotinib for CCyR or MMR rates for 12 months' follow-up (CCyR, odds ratio 1.09, 95% CI 0.61 to 1.92; MMR, odds ratio 1.28, 95% CI 0.77 to 2.16). There is observational association evidence from imatinib studies supporting the use of CCyR and MMR at 12 months as surrogates for overall all-cause survival and progression-free survival in patients with CML in chronic phase. In the cost-effectiveness modelling scenario, analyses were provided to reflect the extensive structural uncertainty and different approaches to estimating OS. First-line dasatinib is predicted to provide very poor value for money compared with first-line imatinib, with deterministic incremental cost-effectiveness ratios (ICERs) of between £256,000 and £450,000 per quality-adjusted life-year (QALY). Conversely, first-line nilotinib provided favourable ICERs at the willingness-to-pay threshold of £20,000-30,000 per QALY. - Limitations Immaturity of empirical trial data relative to life expectancy, forcing either reliance on surrogate relationships or cumulative survival/treatment duration assumptions. - Conclusions From the two trials available, dasatinib and nilotinib have a statistically significant advantage compared with imatinib as measured by MMR or CCyR. Taking into account the treatment pathways for patients with CML, i.e. assuming the use of second-line nilotinib, first-line nilotinib appears to be more cost-effective than first-line imatinib. Dasatinib was not cost-effective if decision thresholds of £20,000 per QALY or £30,000 per QALY were used, compared with imatinib and nilotinib. Uncertainty in the cost-effectiveness analysis would be substantially reduced with better and more UK-specific data on the incidence and cost of stem cell transplantation in patients with chronic CML. - Funding The Health Technology Assessment Programme of the National Institute for Health Research.

Antigen-specific CD4 cells assist CD8 T-effector cells in eliminating keratinocytes

Keratinocytes expressing tumor or viral antigens can be eliminated by antigen-primed CD8 cytotoxic T cells. CD4 T-helper cells help induction of CD8 cytotoxic T cells from naive precursors and generation of CD8 T-cell memory. In this study, we show, unexpectedly, that CD4 cells are also required to assist primed CD8 effector T cells in rejection of skin expressing human growth hormone, a neo-self-antigen, in keratinocytes. The requirement for CD4 cells can be substituted by CD40 costimulation. Rejection of skin expressing ovalbumin (OVA), a non-self-antigen, by primed CD8 cytotoxic T cells can in contrast occur without help from antigen-specific CD4 T cells. However, rejection of OVA expressing keratinocytes is helped by antigen-specific CD4 T cells if only low numbers of primed or naive OVA-specific CD8 T cells are available. Effective immunotherapy directed at antigens expressed in squamous cancer may therefore be facilitated by induction of tumor antigen-specific CD4 helper T cells, as well as cytotoxic CD8 T cells.

A combination of local inflammation and central memory T cells potentiates immunotherapy in the skin

Adoptive T cell therapy uses the specificity of the adaptive immune system to target cancer and virally infected cells. Yet the mechanism and means by which to enhance T cell function are incompletely described, especially in the skin. In this study, we use a murine model of immunotherapy to optimize cell-mediated immunity in the skin. We show that in vitro - derived central but not effector memory-like T cells bring about rapid regression of skin-expressing cognate Ag as a transgene in keratinocytes. Local inflammation induced by the TLR7 receptor agonist imiquimod subtly yet reproducibly decreases time to skin graft rejection elicited by central but not effector memory T cells in an immunodeficient mouse model. Local CCL4, a chemokine liberated by TLR7 agonism, similarly enhances central memory T cell function. In this model, IL-2 facilitates the development in vivo of effector function from central memory but not effector memory T cells. In a model of T cell tolerogenesis, we further show that adoptively transferred central but not effector memory T cells can give rise to successful cutaneous immunity, which is dependent on a local inflammatory cue in the target tissue at the time of adoptive T cell transfer. Thus, adoptive T cell therapy efficacy can be enhanced if CD8+ T cells with a central memory T cell phenotype are transferred, and IL-2 is present with contemporaneous local inflammation. Copyright © 2012 by The American Association of Immunologists, Inc.

Transcriptome and glycome profiling of human hematopoietic CD133+ and CD34+ cells

New blood cells are continuously provided by self-renewing multipotent hematopoietic stem cells (HSC). The capacity of HSCs to regenerate the hematopoietic system is utilized in the treatment of patients with hematological malignancies. HSCs can be enriched using an antibody-based recognition of CD34 or CD133 glycoproteins on the cell surface. The CD133+ and CD34+ cells may have partly different roles in hematopoiesis. Furthermore, each cell has a glycome typical for that cell type. Knowledge of HSC glycobiology can be used to design therapeutic cells with improved cell proliferation or homing properties. The present studies characterize the global gene expression profile of human cord blood-derived CD133+ and CD34+ cells, and demonstrate the differences between CD133+ and CD34+ cell populations that may have an impact in transplantation when CD133+ and CD34+ selected cells are used. In addition, these studies unravel the glycome profile of primitive hematopoietic cells and reveal the transcriptional regulation of N-glycan biosynthesis in CD133+ and CD34+ cells. The gene expression profile of CD133+ cells represents 690 differentially expressed transcripts between CD133+ cells and CD133- cells. CD34+ cells have 620 transcripts differentially expressed when compared to CD34- cells. The integrated CD133+/CD34+ cell gene expression profiles proffer novel transcripts to specify HSCs. Furthermore, the differences between the gene expression profiles of CD133+ and CD34+ cells indicate differences in the transcriptional regulation of CD133+ and CD34+ cells. CD133+ cells express a lower number of hematopoietic lineage differentiation marker genes than CD34+ cells. The expression profiles suggest a more primitive nature of CD133+ cells. Moreover, CD133+ cells have characteristic glycome that differ from the glycome of CD133- cells. High mannose-type and biantennary complex-type N-glycans are enriched in CD133+ cells. N-glycosylation-related gene expression pattern of CD133+ cells identify the key genes regulating the CD133+ cell-specific glycosylation including the overexpression of MGAT2 and underexpression of MGAT4. The putative role of MAN1C1 in the increase of unprocessed high mannose-type N-glycans in CD133+ cells is also discussed. These studies provide new information on the characteristics of HSCs. Improved understanding of HSC biology can be used to design therapeutic cells with improved cell proliferation and homing properties. As a result, HSC engineering could further their clinical use.

Mechanisms and molecular regulation of mammalian tooth replacement

In most non-mammalian vertebrates, such as fish and reptiles, teeth are replaced continuously. However, tooth replacement in most mammals, including human, takes place only once and further renewal is apparently inhibited. It is not known how tooth replacement is genetically regulated, and little is known on the physiological mechanism and evolutionary reduction of tooth replacement in mammals. In this study I have attempted to address these questions. In a rare human condition cleidocranial dysplasia, caused by a mutation in a Runt domain transcription factor Runx2, tooth replacement is continued. Runx2 mutant mice were used to investigate the molecular mechanisms of Runx2 function. Microarray analysis from dissected embryonic day 14 Runx2 mutant and wild type dental mesenchymes revealed many downstream targets of Runx2, which were validated using in situ hybridization and tissue culture methods. Wnt signaling inhibitor Dkk1 was identified as a candidate target, and in tissue culture conditions it was shown that Dkk1 is induced by FGF4 and this induction is Runx2 dependent. These experiments demonstrated a connection between Runx2, FGF and Wnt signaling in tooth development and possibly also in tooth replacement. The role of Wnt signaling in tooth replacement was further investigated by using a transgenic mouse model where Wnt signaling mediator β-catenin is continuously stabilized in dental epithelium. This stabilization led to activated Wnt signaling and to the formation of multiple enamel knots. In vitro and transplantation experiments were performed to examine the process of extra tooth formation. We showed that new teeth were continuously generated and that new teeth form from pre-existing teeth. A morphodynamic activator-inhibitor model was used to simulate enamel knot formation. By increasing the intrinsic production rate of the activator (β-catenin), the multiple enamel knot phenotype was reproduced by computer simulations. It was thus concluded that β-catenin acts as an upstream activator of enamel knots, closely linking Wnt signaling to the regulation of tooth renewal. As mice do not normally replace teeth, we used other model animals to investigate the physiological and genetic mechanisms of tooth replacement. Sorex araneus, the common shrew was earlier reported to have non-functional tooth replacement in all antemolar tooth positions. We showed by histological and gene expression studies that there is tooth replacement only in one position, the premolar 4 and that the deciduous tooth is diminished in size and disappears during embryogenesis without becoming functional. The growth rates of deciduous and permanent premolar 4 were measured and it was shown by competence inference that the early initiation of the replacement tooth in relation to the developmental stage of the deciduous tooth led to the inhibition of deciduous tooth morphogenesis. It was concluded that the evolutionary loss of deciduous teeth may involve the early activation of replacement teeth, which in turn suppress their predecessors. Mustela putorius furo, the ferret, has a dentition that resembles that of the human as ferrets have teeth that belong to all four tooth families, and all the antemolar teeth are replaced once. To investigate the replacement mechanism, histological serial sections from different embryonic stages were analyzed. It was noticed that tooth replacement is a process which involves the growth and detachment of the dental lamina from the lingual cervical loop of the deciduous tooth. Detachment of the deciduous tooth leads to a free successional dental lamina, which grows deeper into the mesenchyme, and later buds the replacement tooth. A careful 3D analysis of serial histological sections was performed and it was shown that replacement teeth are initiated from the successional dental lamina and not from the epithelium of the deciduous tooth. The molecular regulation of tooth replacement was studied and it was shown by examination of expression patterns of candidate regulatory genes that BMP/Wnt inhibitor Sostdc1 was strongly expressed in the buccal aspect of the dental lamina, and in the intersection between the detaching deciduous tooth and the successional dental lamina, suggesting a role for Sostdc1 in the process of detachment. Shh was expressed in the enamel knot and in the inner enamel epithelium in both generations of teeth supporting the view that the morphogenesis of both generations of teeth is regulated by similar mechanisms. In summary, histological and molecular studies on different model animals and transgenic mouse models were used to investigate tooth replacement. This thesis work has significantly contributed to the knowledge on the physiological mechanisms and molecular regulation of tooth replacement and its evolutionary suppression in mammals.

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation Loss of facial symmetry and mimetic function as seen in facial paralysis has an enormous impact on the psychosocial conditions of the patients. Patients with severe long-term facial paralysis are often reanimated with a two-stage procedure combining cross-facial nerve grafting, and 6 to 8 months later with microneurovascular (MNV) muscle transfer. In this thesis, we recorded the long-term results of MNV surgery in facial paralysis and observed the possible contributing factors to final functional and aesthetic outcome after this procedure. Twenty-seven out of forty patients operated on were interviewed, and the functional outcome was graded. Magnetic resonance imaging (MRI) of MNV muscle flaps was done, and nerve graft samples (n=37) were obtained in second stage of the operation and muscle biopsies (n=18) were taken during secondary operations.. The structure of MNV muscles and nerve grafts was evaluated using histological and immunohistochemical methods ( Ki-67, anti-myosin fast, S-100, NF-200, CD-31, p75NGFR, VEGF, Flt-1, Flk-1). Statistical analysis was performed. In our studies, we found that almost two-thirds of the patients achieved good result in facial reanimation. The longer the follow-up time after muscle transfer the weaker was the muscle function. A majority of the patients (78%) defined their quality of life improved after surgery. In MRI study, the free MNV flaps were significantly smaller than originally. A correlation was found between good functional outcome and normal muscle structure in MRI. In muscle biopsies, the mean muscle fiber diameter was diminished to 40% compared to control values. Proliferative activity of satellite cells was seen in 60% of the samples and it tended to decline with an increase of follow-up time. All samples showed intramuscular innervation. Severe muscle atrophy correlated with prolonged intraoperative ischaemia. The good long-term functional outcome correlated with dominance of fast fibers in muscle grafts. In nerve grafts, the mean number of viable axons amounted to 38% of that in control samples. The grafted nerves characterized by fibrosis and regenerated axons were thinner than in control samples although they were well vascularized. A longer time between cross facial nerve grafting and biopsy sampling correlated with a higher number of viable axons. P75Nerve Growth Factor Receptor (p75NGFR) was expressed in every nerve graft sample. The expression of p75NGFR was lower in older than in younger patients. A high expression of p75NGFR was often seen with better function of the transplanted muscle. In grafted nerve Vascular Endothelial Growth Factor (VEGF) and its receptors were expressed in nervous tissue. In conclusion, most of the patients achieved good result in facial reanimation and were satisfied with the functional outcome. The mimic function was poorer in patients with longer follow-up time. MRI can be used to evaluate the structure of the microneurovascular muscle flaps. Regeneration of the muscle flaps was still going on many years after the transplantation and reinnervation was seen in all muscle samples. Grafted nerves were characterized by fibrosis and fewer, thinner axons compared to control nerves although they were well vascularized. P75NGFR and VEGF were expressed in human nerve grafts with higher intensity than in control nerves which is described for the first time.

Cell surface heparan sulfate proteoglycans as novel markers of human neural stem cell fate determination

Multipotent neural stem cells (NSCs) provide a model to investigate neurogenesis and develop mechanisms of cell transplantation. In order to define improved markers of stemness and lineage specificity, we examined self-renewal and multi-lineage markers during long-term expansion and under neuronal and astrocyte differentiating conditions in human ESC-derived NSCs (hNSC H9 cells). In addition, with proteoglycans ubiquitous to the neural niche, we also examined heparan sulfate proteoglycans (HSPGs) and their regulatory enzymes. Our results demonstrate that hNSC H9 cells maintain self-renewal and multipotent capacity during extended culture and express HS biosynthesis enzymes and several HSPG core protein syndecans (SDCs) and glypicans (GPCs) at a high level. In addition, hNSC H9 cells exhibit high neuronal and a restricted glial differentiative potential with lineage differentiation significantly increasing expression of many HS biosynthesis enzymes. Furthermore, neuronal differentiation of the cells upregulated SDC4, GPC1, GPC2, GPC3 and GPC6 expression with increased GPC4 expression observed under astrocyte culture conditions. Finally, downregulation of selected HSPG core proteins altered hNSC H9 cell lineage potential. These findings demonstrate an involvement for HSPGs in mediating hNSC maintenance and lineage commitment and their potential use as novel markers of hNSC and neural cell lineage specification.

Cytomegalovirus in the Process of Chronic Allograft Nephropathy

Cytomegalovirus (CMV) is a major cause of morbidity, costs and even mortality in organ transplant recipients. CMV may also enhance the development of chronic allograft nephropathy (CAN), which is the most important cause of graft loss after kidney transplantation. The evidence for the role of CMV in chronic allograft nephropathy is somewhat limited, and controversial results have also been reported. The aim of this study was to investigate the role of CMV in the pathogenesis of CAN. Material for the purpose of this study was available from altogether 70 kidney transplant recipients who received a kidney transplant between the years 1992-2000. CMV infection was diagnosed with pp65 antigenemia test or by viral culture from blood, urine, or both. CMV proteins were demonstrated in the kidney allograft biopsies by immunohistochemisrty and CMV-DNA by in situ hybridization. Cytokines, adhesion molecules, and growth factors were demonstrated from allograft biopsies by immunohistochemistry, and from urinary samples by ELISA-methods. CMV proteins were detectable in the 6-month protocol biopsies from 18/41 recipients with evidence of CMV infection. In the histopathological analysis of the 6-month protocol biopsies, presence of CMV in the allograft together with a previous history of acute rejection episodes was associated with increased arteriosclerotic changes in small arterioles. In urinary samples collected during CMV infection, excretion of TGF-β was significantly increased. In recipients with increased urinary excretion of TGF-β, increased interstitial fibrosis was recorded in the 6- month protocol biopsies. In biopsies taken after an active CMV infection, CMV persisted in the kidney allograft in 17/48 recipients, as CMV DNA or antigens were detected in the biopsies more than 2 months after the last positive finding in blood or urine. This persistence was associated with increased expression of TGF-β, PDGF, and ICAM-1 and with increased vascular changes in the allografts. Graft survival and graft function one and two years after transplantation were reduced in recipients with persistent intragraft CMV. Persistent intragraft CMV infection was also a risk factor for reduced graft survival in Cox regression analysis, and an independent risk factor for poor graft function one and two years after transplantation in logistic regression analysis. In conclusion, these results show that persistent intragraft CMV infection is detrimental to kidney allografts, causing increased expression of growth factors and increased vascular changes, leading to reduced graft function and survival. Effective prevention, diagnosis and treatment of CMV infections may a major factor in improving the long term survival of kidney allograft.