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Protocol biopsies are used to monitor allograft histology after transplantation. However, biopsy is an invasive procedure with potential complications, requires special facilities, and is unpractical for repeated monitoring of the graft. A noninvasive, robust, and rapid diagnostic method would be welcomed. Monitoring gene expression from blood samples could provide such a means.

Cord blood is a rich source of stem cells especially for hematopoietic stem cells. Recently, mesenchymal stem cells (MSCs) have also been shown to exist in cord blood. However, these fibroblast‐like multipotent progenitor cells are rather rare in cord blood. Many different methods have been used for their culture. This unit describes one method to obtain MSCs from cord blood and another method to differentiate these cells into osteoblasts, which is one of the lineages that mesenchymal stem cells are capable of differentiating into. The starting material for the protocol is cord blood–derived mononuclear cells. As cord blood contains a great number of erythroid precursors, the glycophorin A–positive cells are depleted using magnetic cell separation to reduce their presence in MSC culture. Osteoblast differentiation and a method to demonstrate the result of the differentiation are also described in this unit. Curr. Protoc. Stem Cell Biol. 1:2A.3.1‐2A.3.7. © 2007 by John Wiley & Sons, Inc.

Enrichment of hematopoietic stem cells is based on the expression of certain surface antigens, such as CD34 and CD133, or on the lack of expression of lineage‐specific antigens. Immunomagnetic positive selection of CD34+or CD133+cells is performed using paramagnetic microbeads conjugated to specific monoclonal antibodies (anti–human CD34 or anti–human CD133). In negative selection of lineage‐negative (Lin−) cells, the unwanted cells are labeled with antibodies against known markers for mature hematopoietic cells (CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and glycophorin A) and retained in the column. Unlabeled cells pass through the column and are collected as the Lin−cell fraction. Immunomagnetic cell sorting system MACS is a fast and gentle method to enrich hematopoietic stem cells. Viable and highly pure cells can be separated to be used in various downstream applications, such as flow cytometry and cell culture. Curr. Protoc. Stem Cell Biol. 1:2A.2.1‐2A.2.9. © 2007 by John Wiley & Sons, Inc.

When preparing stem cell specimens from cord blood, pre‐enrichment of mononuclear cells is highly recommended to improve the recovery of rare stem cells. Mononuclear cells are easily isolated by density gradient centrifugation. In Ficoll‐Paque density gradient centrifugation, anticoagulant‐treated and diluted cord blood is layered on the Ficoll‐Paque solution and centrifuged. During centrifugation, erythrocytes and granulocytes sediment to the bottom layer. Lower density lymphocytes, together with other slowly sedimenting cells such as platelets and monocytes, are retained at the interface between the plasma and Ficoll‐Paque, where they can be collected and subjected to subsequent isolation of hematopoietic stem cells or to the culture of mesenchymal stem cells. Curr. Protoc. Stem Cell Biol. 1:2A.1.1‐2A.1.4. © 2007 by John Wiley & Sons, Inc.

Currently, the diagnosis of acute rejection after kidney transplantation is based on a kidney biopsy taken after clinical rejection suspicion. A robust, noninvasive diagnostic method would allow easier and more frequent monitoring of the patient and the graft. Potentially, a straightforward method would be the analysis of lymphocyte marker molecule expression from whole blood samples.

CD34 and CD133 are the most commonly used markers to enrich hematopoietic stem cells (HSCs). Positively selected HSCs are increasingly used for autologous and allogeneic transplantation, yet the biological properties of CD34+ and CD133+ cells are largely unknown. In the present study, a genome-wide gene expression analysis of human cord blood (CB)-derived CD34+ cells was performed. The CD34+ gene expression profile was compared to an identically constructed CD133+ gene expression profile to reveal the specific expression patterns and major differences of CD34+ and CD133+ cells. As expected, many genes were similarly expressed in the two cell populations, but cell-type-specific gene expression was also demonstrated. Self-organizing map analysis was used to identify transcripts having similar expression patterns, and the results were compared between CD34+ and CD133+ cells. Also, a prioritization algorithm was used to rank the genes best separating CD34+ and CD133+ cells from their CD34− and CD133− counterparts in CB. Our results show that CD133+ cells have higher numbers of up-regulated genes than CD34+ cells. Furthermore, the uniquely expressed genes in CD34+ or CD133+ cell populations were associated with different biological processes. CD34+ cells overexpressed many transcripts associated with development and response to stress or external stimuli. In CD133+ cells, the most significantly represented biological processes were establishment and maintenance of chromatin architecture, DNA metabolism, and cell cycle. The differences between the gene expression profiles of CD34+ and CD133+ cells indicate the more primitive nature of CD133+ cells. These profiles suggest that CD34+ and CD133+ cells may have different roles in hematopoietic regeneration.

Pretransplantation identification of patients at an increased risk for adverse events would allow more individualized treatment strategies possibly improving long-term outcome. We studied cytokine gene polymorphisms of kidney allograft recipients and their donors to identify factors predisposing for acute rejection (AR) and delayed graft function (DGF).

ABSTRACTSerine/threonine kinase Akt/protein kinase B, the cellular homologue of the transforming viral oncogene v-Akt, plays a central role in the regulation of cell survival and proliferation. We have previously demonstrated that the proto-oncogene TCL1 is an Akt kinase coactivator. TCL1 binds to Akt and mediates the formation of oligomeric TCL1-Akt high-molecular-weight protein complexes in vivo. Within these protein complexes, Akt is preferentially phosphorylated and activated. The MTCP1/TCL1/TCL1b oncogene activation is the hallmark of human T-cell prolymphocytic leukemia (T-PLL), a form of adult leukemia. In the present study, using a PCR-generated random TCL1 library combined with a yeast two-hybrid screening detecting loss of interaction, we identified D16 and I74 as amino acid residues mediating the association of TCL1 with Akt. Based on molecular modeling, we determined that the βC-sheet of TCL1 is essential for TCL1 homodimerization. Studies with mammalian overexpression systems demonstrated that both Akt association and oligomerization domains of TCL1 are distinct functional domains. In vitro kinase assays and overexpression experiments in mammalian cells demonstrated that both TCL1-Akt interaction and oligomerization of TCL1 were required for TCL1-induced Akt activation and substrate phosphorylation. Assays for mitochondrial permeability transition, nuclear translocation, and cell recovery demonstrated that both Akt association and homodimerization of TCL1 are similarly needed for the full function of TCL1 as an Akt kinase coactivator in vivo. The results demonstrate the structural basis of TCL1-induced activation of Akt, which causes human T-PLL.

Serine/threonine kinase Akt/protein kinase B, the cellular homologue of the transforming viral oncogene v-Akt, plays a central role in the regulation of cell survival and proliferation. We have previously demonstrated that the proto-oncogene TCL1 is an Akt kinase coactivator. TCL1 binds to Akt and mediates the formation of oligomeric TCL1-Akt high-molecular-weight protein complexes in vivo. Within these protein complexes, Akt is preferentially phosphorylated and activated. The MTCP1/TCL1/TCL1b oncogene activation is the hallmark of human T-cell prolymphocytic leukemia (T-PLL), a form of adult leukemia. In the present study, using a PCR-generated random TCL1 library combined with a yeast two-hybrid screening detecting loss of interaction, we identified D16 and I74 as amino acid residues mediating the association of TCL1 with Akt. Based on molecular modeling, we determined that the βC-sheet of TCL1 is essential for TCL1 homodimerization. Studies with mammalian overexpression systems demonstrated that both Akt association and oligomerization domains of TCL1 are distinct functional domains. In vitro kinase assays and overexpression experiments in mammalian cells demonstrated that both TCL1-Akt interaction and oligomerization of TCL1 were required for TCL1-induced Akt activation and substrate phosphorylation. Assays for mitochondrial permeability transition, nuclear translocation, and cell recovery demonstrated that both Akt association and homodimerization of TCL1 are similarly needed for the full function of TCL1 as an Akt kinase coactivator in vivo. The results demonstrate the structural basis of TCL1-induced activation of Akt, which causes human T-PLL.

The members of the TCL1 proto-oncogene family (TCL1, MTCP1, and TCL1b) bind to Akt1, increasing its phosphorylation status and kinase activity. This is thought to be secondary to the formation of TCL1-Akt oligomers within which Akt is preferentially phosphorylated. Here we show that, in contrast to Akt1 and Akt2, which bind to all members of the TCL1 family, Akt3 specifically interacts with TCL1 but not with MTCP1 or TCL1b. This association is functional, as the presence of TCL1 but not MTCP1 or TCL1b increased Akt3 kinase activity in in vitrokinase assays. Functional specificity is determined by the Akt pleckstrin homology domain as chimeric Akt1, where Akt1 PH domain was replaced by that of Akt3 was no longer able to interact with MTCP1 or TCL1b and its kinase activity was solely enhanced by TCL1. Moreover, we show that, in TCL1-overexpressing SUPT-11 T-cell leukemia and P3HR-1 Burkitt's lymphoma cell lines, TCL1 interacts with endogenous Akt1, Akt2, and Akt3. TCL1 enhanced hetero-oligomerization of Akt1 with Akt3 and as a consequence facilitated transphosphorylation of Akt molecules, which may contribute to Akt activation and TCL1-induced leukemogenesis in vivo.

Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1) is caused by mutations in the NPHS1gene. NPHS1codes for nephrin, a cell adhesion protein located at the glomerular slit diaphragm. Renal transplantation is the only treatment option for most patients with NPHS1. We have previously described recurrence of severe proteinuria in grafts transplanted to children with NPHS1. Here we studied the pathophysiology of this proteinuria.

We report a long-term prospective follow-up of renal allograft histology in children <5 years of age at transplantation (Tx).

Low recipient age is still a risk factor for graft failure after kidney transplantation (Tx). Detailed prospective reports on long-term graft function in small children after renal Tx are still lacking.

The prognosis for primary epithelial liver tumor in children in whom radical surgery cannot be performed after chemotherapy is poor. Orthotopic liver transplantation has resulted in mortality up to 50%, largely as a result of problems in determining the criteria for transplantation.

.Renal transplantation is currently standard therapy for end-stage kidney disease for children. Despite the considerable improvement in short-term results, the expected allograft half-life has remained the same. This is due to chronic rejection/late graft dysfunction which has proved resistant to therapeutic attempts. During the last few years the multifactorial pathogenesis of chronic renal allograft rejection has been clarified to some extent. Early injury by immunological and non-immunological mechanisms is followed by vascular remodelling due to repetitive cycles of cytokine release, upregulation of growth factors, and vascular smooth muscle cell proliferation. This leads to typical concentric arteriolosclerosis and ischemia. Secondary kidney-specific mechanisms are initiated by the reduction in functioning renal mass and lead to gradual progression of chronic rejection. There is no single optimal therapy. Several attempts to influence the pathophysiological cascade have been promising. Attention should be focused on minimizing early immunological/non-immunological injury in order to attenuate future progression of chronic rejection. A significant prolongation of allograft half-life may be achieved during the next decade with the introduction of new therapeutic agents and comprehensive approach to treatment. This would be especially beneficial for pediatric recipients, reducing the need for retransplantation in adulthood.

Renal transplantation is the optimal form of renal replacement therapy leading to substantial improvement in the quality of life. It has rapidly become the standard treatment for end-stage renal disease in children. However, despite impressive short-term results significant long-term problems remain unsolved. Because of the lack of effective treatment for chronic rejection and common recipient noncompliance, allograft half-life has not improved significantly during the last decade. A paediatric recipient is likely to need several retransplantations in adulthood. Moreover, the immunosuppressive drugs used today have potentially serious side-effects including nephrotoxicity and de novo malignancy. These are especially relevant for paediatric recipients who will continue to receive therapy for several decades. Most therapeutic protocols used for children are derived from those used for adults. However, the metabolic differences between an adult and a growing and developing paediatric transplant recipient are not always adequately appreciated before these new therapies are initiated. In the near future, we are likely to see new and more efficient drugs become available. It is important that we try to understand their properties in children and use them and our current arsenal on an individual basis aiming at optimal graft survival but also at avoiding unnecessary adverse effects.

We studied 22 children (mean age: 8.42 years, range: 1.9-15.6 years) with a liver transplant to compare the pharmacokinetics of oral cyclosporine(CsA) microemulsion to the conventional formulation. The CsA treatment (mean dose: 5.9 mg/kg/day, range: 3.0-11.7 mg/kg/day) was converted 1:1 on a milligram-to-milligram basis to the microemulsion formulation. Five days after the conversion, the mean peak blood CsA concentration was higher(microemulsion: 963 ng/ml, range: 518-1864 ng/ml; conventional: 431 ng/ml, range: 98-888 ng/ml; P<0.0001) and it was reached faster (median time of peak concentration: 1.6 hr vs. 2.9 hr, range: 1.0-3.0 hr vs. 1.9-4.0 hr; P=0.0009). The absorption lasted on the average 19% longer after the conventional formulation. The area under the concentration versus time curve (AUC) was larger after the microemulsion formulation in all but one patient (P=0.001) by a mean factor of 1.80 (range: 0.72-3.04). The trough CsA level after the microemulsion formulation was more closely related to peak concentration(r2=0.86 vs. 0.45) and AUC(r2=0.84 vs. 0.47); thus, therapeutic drug monitoring may be more useful. After 6 months on the new formulation, the results for the whole group were similar, but in five children the AUC was comparable to the AUC obtained with the conventional formulation. No rejections occurred, and the liver and kidney functions remained unchanged. A 1:1 conversion can be safely performed in children, based on a 6-month follow-up. However, the total drug exposure changes in significant ways, which, on a long-term basis, may improve the immunosuppression in an underimmunosuppressed patient, but may increase the risk for dose-related adverse effects in others.

Linear growth is more often impaired after liver than after renal transplantation (Tx) in childhood. As similar triple immunosuppression was used in our liver and renal transplant recipients, we were able to compare growth and endocrine function between 19 prepubertal liver and 35 renal transplant recipients. There were no significant differences in median age, weight-for-height index, or height standard deviation score at Tx. Seventy-eight percent of the liver Tx patients, but only 7 of the renal Tx patients, were below the normal range for height 3 years after Tx. Graft function was good in both liver and renal transplant recipients 3 years after Tx. There was no significant difference in growth hormone secretion, serum insulin- like growth factor (IGF)-I, and IGF-binding protein- 3 levels, or in methylprednisolone and cyclosporine doses. However, the blood cyclosporine levels were significantly higher in the liver transplant recipients (P0.001 1 year and P0.005 2 years after Tx). Cortisol production was significantly lower in the liver transplant recipients (P0.002 1 year and P0.049 2 years after Tx), which suggests greater steroid- mediated suppression of adrenal function.

Alphacalcidol oral pulse therapy was given for secondary hyperparathyroidism to 22 children (mean age of 5.6 years) with renal insufficiency. At the beginning of the study, the glomerular filtration rate was <50% of normal, serum intact parathyroid hormone (PTH) was >100 ng/l and the serum phosphate and calcium concentrations were within the normal range. Alphacalcidol (0.5–3.0 µg) was given orally thrice weekly in the evening and adjusted according to PTH, ionized calcium and phosphate concentrations. Serum PTH (mean ± SEM) decreased significantly from a pre-treatment level of 393±81 ng/l to 122±34 ng/l after 12 months, and stabilized at this level. Mean vitamin D metabolite concentrations were within the normal range. 1,25-Dihydroxyvitamin D did not increase during therapy, while PTH decreased. The estimated creatinine clearance remained almost unchanged (20±3 and 21±6 ml/min per 1.73 m2). Growth remained low normal (height standard deviation score -1.8±0.3 initially and -1.7±0.4 12 months later) and bone mineral density did not decrease. We concluded that feedback regulation of PTH with oral alphacalcidol pulse therapy is effective in the treatment of hyperparathyroidism in children with renal failure prior to dialysis.

ABSTRACT: Type I tyrosinemia (HTI) is an autosomally recessively inherited disease caused by deficiency of fumarylacetoacetate hydrolase. The disease manifests with liver failure, renal tubular defects, and neurologic crises. Currently orthotopic liver transplantation (OLT) enables patients to survive. However, renal fumarylacetoacetate hydrolase deficiency is not corrected by OLT, and the long-term prognosis of the nephropathy is not known. We investigated tyrosine metabolism, GFR, renal tubular function, and histopathology before and 18-36 mo after OLT in eight patients with HTI. Progressive renal dysfunction was not documented despite continuing, although diminished, urinary succinylacetone excretion in all patients. The mean GFR was 82 mL/min/1.73 m2before and 102 mL at 18 mo and 93 mL at 36 mo after OLT. All patients showed tubular dysfunction before OLT. At 18 mo, glucosuria occurred in one, amino aciduria and phosphaturia in three, and hypercalciuria in six patients. Only hypercalciuria was seen at 36 mo. Renal biopsies showed mild nonspecific changes caused either by minimal progression of the renal disease or by mild cyclosporine nephrotoxicity. In conclusion, patients with HTI had normal GFR, but showed signs of tubular dysfunction 18–36 mo after OLT. Renal function and histopathology should be monitored after OLT for HTI.

Alzheimer's disease (AD) is the most common cause of dementia affecting 36 million people worldwide. As the demographic transition in the developed countries progresses towards older population, the worsening ratio of workers per retirees and the growing number of patients with age-related illnesses such as AD will challenge the current healthcare systems and national economies. For these reasons AD has been identified as a health priority, and various methods for diagnosis and many candidates for therapies are under intense research. Even though there is currently no cure for AD, its effects can be managed. Today the significance of early and precise diagnosis of AD is emphasized in order to minimize its irreversible effects on the nervous system. When new drugs and therapies enter the market it is also vital to effectively identify the right candidates to benefit from these. The main objective of the PredictAD project was to find and integrate efficient biomarkers from heterogeneous patient data to make early diagnosis and to monitor the progress of AD in a more efficient, reliable and objective manner. The project focused on discovering biomarkers from biomolecular data, electrophysiological measurements of the brain and structural, functional and molecular brain images. We also designed and built a statistical model and a framework for exploiting these biomarkers with other available patient history and background data. We were able to discover several potential novel biomarker candidates and implement the framework in software. The results are currently used in several research projects, licensed to commercial use and being tested for clinical use in several trials.