Electric Field Navigated 1-Hz rTMS for Poststroke Motor Recovery: The E-FIT Randomized Controlled Trial.

BACKGROUND: To determine if low-frequency repetitive transcranial magnetic stimulation targeting the primary motor cortex contralateral (M1CL) to the affected corticospinal tract in patients with hemiparetic stroke augments intensive training-related clinical improvement; an extension of the NICHE trial (Navigated Inhibitory rTMS to Contralesional Hemisphere Trial) using an alternative sham coil. METHODS: The present E-FIT trial (Electric Field Navigated 1Hz rTMS for Post-stroke Motor Recovery Trial) included 5 of 12 NICHE trial outpatient US rehabilitation centers. The stimulation protocol remained identical (1 Hz repetitive transcranial magnetic stimulation, M1CL, preceding 60-minute therapy, 18 sessions/6 wks; parallel arm randomized clinical trial). The sham coil appearance mimicked the active coil but without the weak electric field in the NICHE trial sham coil. Outcomes measured 1 week, and 1, 3, and 6 months after the end of treatment included the following: upper extremity Fugl-Meyer (primary, 6 months after end of treatment), Action Research Arm Test, National Institutes of Health Stroke Scale, quality of life (EQ-5D), and safety. RESULTS: Of 60 participants randomized, 58 completed treatment and were included for analysis. Bayesian analysis of combined data from the E-FIT and the NICHE trials indicated that active treatment was not superior to sham at the primary end point (posterior mean odds ratio of 1.94 [96% credible interval of 0.61-4.80]). For the E-FIT intent-to-treat population, upper extremity Fugl-Meyer improvement ≥5 pts occurred in 60% (18/30) active group and 50% (14/28) sham group. Participants enrolled 3 to 6 months following stroke had a 67% (31%-91% CI) response rate in the active group at the 6-month end point versus 50% in the sham group (21.5%-78.5% CI). There were significant improvements from baseline to 6 months for both active and sham groups in upper extremity Fugl-Meyer, Action Research Arm Test, and EQ-5D (P<0.05). Improvement in National Institutes of Health Stroke Scale was observed only in the active group (P=0.004). Ten serious unrelated adverse events occurred (4 active group, 6 sham group, P=0.72). CONCLUSIONS: Intensive motor rehabilitation 3 to 12 months after stroke improved clinical impairment, function, and quality of life; however, 1 Hz-repetitive transcranial magnetic stimulation was not an effective treatment adjuvant in the present sample population with mixed lesion location and extent. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03010462.

Randomized Sham-Controlled Trial of Navigated Repetitive Transcranial Magnetic Stimulation for Motor Recovery in Stroke.

Background and Purpose- We aimed to determine whether low-frequency electric field navigated repetitive transcranial magnetic stimulation to noninjured motor cortex versus sham repetitive transcranial magnetic stimulation avoiding motor cortex could improve arm motor function in hemiplegic stroke patients when combined with motor training. Methods- Twelve outpatient US rehabilitation centers enrolled participants between May 2014 and December 2015. We delivered 1 Hz active or sham repetitive transcranial magnetic stimulation to noninjured motor cortex before each of eighteen 60-minute therapy sessions over a 6-week period, with outcomes measured at 1 week and 1, 3, and 6 months after end of treatment. The primary end point was the percentage of participants improving ≥5 points on upper extremity Fugl-Meyer score 6 months after end of treatment. Secondary analyses assessed changes on the upper extremity Fugl-Meyer and Action Research Arm Test and Wolf Motor Function Test and safety. Results- Of 199 participants, 167 completed treatment and follow-up because of early discontinuation of data collection. Upper extremity Fugl-Meyer gains were significant for experimental ( P<0.001) and sham groups ( P<0.001). Sixty-seven percent of the experimental group (95% CI, 58%-75%) and 65% of sham group (95% CI, 52%-76%) improved ≥5 points on 6-month upper extremity Fugl-Meyer ( P=0.76). There was also no difference between experimental and sham groups in the Action Research Arm Test ( P=0.80) or the Wolf Motor Function Test ( P=0.55). A total of 26 serious adverse events occurred in 18 participants, with none related to the study or device, and with no difference between groups. Conclusions- Among patients 3 to 12 months poststroke, goal-oriented motor rehabilitation improved motor function 6 months after end of treatment. There was no difference between the active and sham repetitive transcranial magnetic stimulation trial arms. Clinical Trial Registration- URL: https://www.clinicaltrials.gov . Unique identifier: NCT02089464.

Navigated Transcranial Magnetic Stimulation: A Biologically Based Assay of Lower Extremity Impairment and Gait Velocity.

Objectives. (a) To determine associations among motor evoked potential (MEP) amplitude, MEP latency, lower extremity (LE) impairment, and gait velocity and (b) determine the association between the presence of a detectable MEP signal with LE impairment and with gait velocity. Method. 35 subjects with chronic, stable LE hemiparesis were undergone TMS, the LE section of the Fugl-Meyer Impairment Scale (LE FM), and 10-meter walk test. We recorded presence, amplitude, and latency of MEPs in the affected tibialis anterior (TA) and soleus (SO). Results. MEP presence was associated with higher LEFM scores in both the TA and SO. MEP latency was larger in subjects with lower LEFM and difficulty walking. Conclusion. MEP latency appears to be an indicator of LE impairment and gait. Significance. Our results support the precept of using TMS, particularly MEP latency, as an adjunctive LE outcome measurement and prognostic technique.

Human CMP-N-acetylneuraminic acid hydroxylase is a novel stem cell marker linked to stem cell-specific mechanisms.

Human stem cells contain substantial amounts of the xenoantigen N-glycolylneuraminic acid (Neu5Gc), although the levels of Neu5Gc are low or undetectable in human body fluids and most other human tissues. The lack of Neu5Gc in human tissues has been previously explained by the loss of hydroxylase activity of the human CMP-N-acetylneuraminic acid hydroxylase (CMAH) protein caused by a genetic error in the human Cmah gene. We thus wanted to investigate whether the human redundant Cmah gene could still function in stem cell-specific processes. In this study, we show that CMAH gene expression is significantly upregulated in the adult stem cell populations studied, both of hematopoietic and mesenchymal origin, and identify CMAH as a novel stem cell marker. The CMAH content co-occurs with higher levels of Neu5Gc within stem cells as measured by mass spectrometric profiling. It seems that despite being enzymatically inactive, human CMAH may upregulate the Neu5Gc content of cells by enhancing Neu5Gc uptake from exogenous sources. Furthermore, exposure to exogenous Neu5Gc caused rapid phosphorylation of beta-catenin in both CMAH overexpressing cells and bone marrow-derived mesenchymal stem cells, thereby inactivating Wnt/beta-catenin signaling. The data demonstrate the first molecular evidence for xenoantigen Neu5Gc-induced alteration of crucial stem cell-specific signaling systems for the maintenance of self renewal. These results add further emphasis to the crucial need for completely xenofree culturing conditions for human stem cells.

The N-glycome of human embryonic stem cells.

BACKGROUND: Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses. RESULTS: The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Lex and H type 2 antennae in sialylated complex-type N-glycans. CONCLUSION: The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.

Glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage.

Human mesenchymal stem cells (MSCs) are adult multipotent progenitor cells. They hold an enormous therapeutic potential, but at the moment there is little information on the properties of MSCs, including their surface structures. In the present study, we analyzed the mesenchymal stem cell glycome by using mass spectrometric profiling as well as a panel of glycan binding proteins. Structural verifications were obtained by nuclear magnetic resonance spectroscopy, mass spectrometric fragmentation, and glycosidase digestions. The MSC glycome was compared to the glycome of corresponding osteogenically differentiated cells. More than one hundred glycan signals were detected in mesenchymal stem cells and osteoblasts differentiated from them. The glycan profiles of MSCs and osteoblasts were consistently different in biological replicates, indicating that stem cells and osteoblasts have characteristic glycosylation features. Glycosylation features associated with MSCs rather than differentiated cells included high-mannose type N-glycans, linear poly-N-acetyllactosamine chains and alpha2-3-sialylation. Mesenchymal stem cells expressed SSEA-4 and sialyl Lewis x epitopes. Characteristic glycosylation features that appeared in differentiated osteoblasts included abundant sulfate ester modifications. The results show that glycosylation analysis can be used to evaluate MSC differentiation state.

N-glycan structures and associated gene expression reflect the characteristic N-glycosylation pattern of human hematopoietic stem and progenitor cells.

OBJECTIVE: Cell surface glycans contribute to the adhesion capacity of cells and are essential in cellular signal transduction. Yet, the glycosylation of hematopoietic stem and progenitor cells (HSPC), such as CD133+ cells, is poorly explored. MATERIALS AND METHODS: N-glycan structures of cord blood-derived CD133+ and CD133- cells were analyzed with mass spectrometric profiling and exoglycosidase digestion, cell surface glycan epitopes with lectin binding assay, and expression of N-glycan biosynthesis-related genes with microarray analysis. RESULTS: Over 10% difference was demonstrated in the N-glycan profiles of CD133+ and CD133- cells. Biantennary complex-type N-glycans were enriched in CD133+ cells. Of the genes regulating the synthesis of these structures, CD133+ cells overexpressed MGAT2 and underexpressed MGAT4. Moreover, the amount of high-mannose type N-glycans and terminal alpha2,3-sialylation was increased in CD133+ cells. Elevated alpha2,3-sialylation was supported by the overexpression of ST3GAL6. CONCLUSION: Our work presents new information on the characters of HSPCs. The new knowledge of HSPC-specific N-glycosylation advances their identification and provides tools to promote HSPC homing and mobilization or targeting to specific tissues.

Diagnosis of acute renal allograft rejection by analyzing whole blood mRNA expression of lymphocyte marker molecules.

BACKGROUND: 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. METHODS: Whole blood samples were collected prospectively in a single kidney transplantation center from 50 adult kidney recipients transplanted between 2001 and 2005. The mRNA expression of granzyme B, perforin, FasL, granulysin, CD154, ICOS, CTLA4 and PD-1 were analyzed with real-time quantitative polymerase chain reaction. RESULTS: The expression of ICOS and CD154 were significantly lower in rejection patients than in control patients (P<0.001). Both genes gave statistically significant area under receiver operating characteristic curve (AUC; 0.87, 0.88) with 84% sensitivity and 100% specificity for CD154 and 76% and 86% for ICOS, respectively. In paired rejection and postrejection therapy samples, the expression of both genes significantly increased during rejection therapy (P<0.001). When rejection patients were compared to patients biopsied because of other reasons of graft dysfunction, both CD154 and ICOS were lower in rejection patients but only CD154 was statistically significant (P=0.028, AUC=0.740, sensitivity 52%, specificity 90%). The other studied genes gave no consistent statistically significant results. CONCLUSIONS: The whole blood gene expression quantities of costimulatory molecules CD154 and ICOS reasonably robustly differentiated rejection patients from control patients. The clinical use of the analysis is limited by poor capability to differentiate patients with rejection from patients with other causes of graft dysfunction.

Identification of Akt association and oligomerization domains of the Akt kinase coactivator TCL1.

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 beta C-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.

Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells.

BACKGROUND: There is a growing interest in cord blood as a source of primitive stem cells with the capacity for multilineage differentiation. Pure cell fractions are needed for the characterization and in vitro expansion of stem cells as well as for their use in preclinical research. However, enrichment of stem cells is challenging due to the lack of stem cell-specific markers and gentle protocols for the isolation of highly pure stem cell fractions. Protocols developed for the enrichment of peripheral blood-derived stem cells have been found to be suboptimal for cord blood. RESULTS: In this study, immunomagnetic cell sorting protocols to purify CD34+, CD133+ and Lin- cells from fresh and cryopreserved cord blood were optimized. Reproducible purities of up to 97% were reached. The selected cells were highly viable having substantial colony-forming potential. CONCLUSION: The optimized protocols enable rapid enrichment of highly pure hematopoietic stem cells from both fresh and cryopreserved cord blood.

Transcriptional profiling reflects shared and unique characters for CD34+ and CD133+ cells.

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.

The impact of donor cytokine gene polymorphisms on the incidence of cytomegalovirus infection after kidney transplantation.

Cytomegalovirus (CMV) seronegative recipients of kidneys from CMV seropositive donors are at a high risk of CMV infection after transplantation since viruses in the allograft may reactivate in patients without prior immunity. We hypothesized that the genetic background of the graft has an influence on the incidence of infection. Effects of IL10, IL6 and IFNG gene polymorphisms, known to affect CMV infectivity, were investigated in 71 CMV seronegative recipients of grafts from CMV seropositive cadaver donors. Donor IL10(-1082 AA) genotype reduced the incidence of CMV infection (p=0.031) and CMV episodes in these patients tended to occur later (AA: median 83 days, AG/GG: median 45 days, p=0.072). In multivariate analysis, other explaining factors than the donor IL10(-1082 AA) genotype alone did not improve Cox hazard model (HR=0.3, 95% CI=0.09-0.96, p=0.043). Recipient polymorphisms did not reduce the incidence of CMV infection. We conclude that donor IL10 gene polymorphisms may influence the likelihood of CMV infection in the high risk patients investigated.

Human umbilical cord blood cells do not improve sensorimotor or cognitive outcome following transient middle cerebral artery occlusion in rats.

The present study investigated effects of human umbilical cord blood (HUCB) cells on sensorimotor, cognitive, and histological outcome in rats subjected to transient middle cerebral artery occlusion (MCAO). Halothane anesthetized adult male Wistar rats were subjected to transient MCAO for 2 h. HUCB cells (mononuclear 1-5x10(7) or Lin(-) cells 1-5x10(5)) were administered intravenously after 24 h recovery. The limb-placing test was performed on postoperative days 2, 4, 6, 9, 12, 16, and 20. In addition, beam-walking and cylinder tests were used to assess sensorimotor function at baseline, and on postoperative days 4, 12, and 20. Morris water-maze was used to assess cognitive performance on postoperative days 22-24. Subsequently, rats were perfused for measurement of infarct volumes and detection of HUCB cells by immunohistochemistry (MAB1281). MCAO rats showed a partial spontaneous recovery in sensorimotor function during the follow-up. However, the recovery profile was similar in MCAO controls and in MCAO rats that received HUCB cells. HUCB did not affect impaired water-maze performance of MCAO rats. Only few human nuclei-specific MAB1281-positive cells were detected in the ipsilateral hemisphere in MCAO rats that received HUCB cells. Infarct volumes did not differ between the experimental groups. A group of additional rats were used to further study biodistribution of intravenously given (111)In-oxine-labelled mononuclear HUCB cells in MCAO and sham-operated rats. SPECT imaging data indicated a high tracer uptake in the lung, liver, spleen, and kidney, but not in the brain immediately after administration or 24 h post-administration. The present study suggests that HUCB cells do not improve functional recovery or histological outcome in MCAO rats after systemic administration because of limited migration of cells in the ischemic brain.

Human cord blood CD34+ cells and behavioral recovery following focal cerebral ischemia in rats.

The present study investigated effects of human umbilical cord blood derived CD34+ cells on sensorimotor, cognitive, and histological outcome in rats following focal cerebral ischemia. Halothane anesthetized adult male Wistar rats were subjected to transient or permanent occlusion of the middle cerebral artery (MCAO) followed by intravenous administration of CD34+ cells (5 x 10(5) or 2 x 10(6)) after 24 h recovery. The beam-walking and cylinder tests were used to assess sensorimotor function, and Morris water-maze examined cognitive performance during a 25 day follow-up period. Subsequently, rats were perfused for measurement of infarct volumes and detection of CD34' cells in the brain by immunohistochemistry (MAB1281). MCAO rats showed minor or no spontaneous recovery in sensorimotor function during the follow-up. The recovery profile was similar in MCAO controls and in MCAO rats that received CD34+ cells, although CD34+ cells seemed to improve the use of impaired forelimb. There was also a trend toward improved water-maze performance by CD34+ cells in transient MCAO rats. Infarct volumes assessed from Nissl-stained sections on postoperative day 25 did not differ between the experimental groups. MAB 1281-positive cells were not detected in the brain of MCAO rats that received CD34+ cells. The present study suggests that CD34+ cells might improve functional outcome in MCAO rats after systemic administration, but do not significantly provide neuroprotection.

Facilitatory effect of paired-pulse stimulation by transcranial magnetic stimulation with biphasic wave-form.

Transcranial magnetic stimulation (TMS) is used to probe corticospinal excitability by stimulating the motor cortex. Our aim was to enhance the effects of biphasic TMS by coupling a suprathreshold test pulse and a following subthreshold priming pulse to induce short-interval intracortical facilitation (SICF), which is conventionally produced with monophasic TMS. Biphasic TMS could potentially induce the SICF effect with better energy-efficiency and with lower stimulus intensities. This would make the biphasic paired-pulses better applicable in patients with reduced cortical excitability. A prototype stimulator was built to produce biphasic paired-pulses. Resting motor thresholds (rMTs) from the right and left hand abductor pollicis brevis muscles, and the right tibialis anterior muscle of eight healthy volunteers were determined using single-pulse paradigm with neuronavigated TMS. The rMTs and MEPs were measured using single-pulses and three paired-pulse setups (interstimulus interval, ISI of 3, 7 or 15ms). The rMTs were lower and MEPs were higher with biphasic paired-pulses compared to single-pulses. The SICF effect was greatest at 3ms ISI. This suggests that the application of biphasic paired-pulses to enhance stimulation effects is possible.

Placenta--an alternative source of stem cells.

The two most promising practical applications of human stem cells are cellular replacement therapies in human disease and toxicological screening of candidate drug molecules. Both require a source of human stem cells that can be isolated, purified, expanded in number and differentiated into the cell type of choice in a controlled manner. Currently, uses of both embryonic and adult stem cells are investigated. While embryonic stem cells are pluripotent and can differentiate into any specialised cell type, their use requires establishment of embryonic stem cell lines using the inner cell mass of an early pre-implantation embryo. As the blastocyst is destroyed during the process, ethical issues need to be carefully considered. The use of embryonic stem cells is also limited by the difficulties in growing large numbers of the cells without inducing spontaneous differentiation, and the problems in controlling directed differentiation of the cells. The use of adult stem cells, typically derived from bone marrow, but also from other tissues, is ethically non-controversial but their differentiation potential is more limited than that of the embryonic stem cells. Since human cord blood, umbilical cord, placenta and amnion are normally discarded at birth, they provide an easily accessible alternative source of stem cells. We review the potential and current status of the use of adult stem cells derived from the placenta or umbilical cord in therapeutic and toxicological applications.

Recurrence of nephrotic syndrome in kidney grafts of patients with congenital nephrotic syndrome of the Finnish type: role of nephrin.

BACKGROUND: Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1) is caused by mutations in the NPHS1 gene. NPHS1 codes 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. METHODS: Clinical data, light and electron microscopic findings as well as the expression of nephrin in the proteinuric grafts were studied. The patients' sera were screened for antibodies against kidney glomerulus and nephrin molecule using indirect immunofluorescence and ELISA. RESULTS: Fifteen episodes of recurrent nephrotic syndrome occurred in 13 (25%) of 51 grafts transplanted to 45 Finnish children with NPHS1. All nine patients with recurrence had a Fin-major/Fin-major genotype, which leads to absence of nephrin in the native kidney. Rescue therapy (cyclophosphamide) was successful in seven episodes, but six kidneys were lost due to this process. Antibodies reacting against glomerulus were found in eight, and high anti-nephrin antibody levels were detected in four of the nine patients. In electron microscopy, the fusion of the foot process and decreases in the detectable slit diaphragms in the podocyte pores were observed. The expression of nephrin mRNA was markedly reduced in two, and granular staining for nephrin was seen in three of five grafts. CONCLUSIONS: Circulating anti-nephrin antibodies seem to have a pathogenic role in the development of heavy proteinuria in kidney grafts of NPHS1 patients with Fin-major/Fin-major genotype.

Cytokine gene polymorphisms and risks of acute rejection and delayed graft function after kidney transplantation.

BACKGROUND: 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). METHODS: A total of 291 adult cadaver kidney recipients transplanted at a single transplantation centre between 1999 and 2002 were investigated. Recipients and donors were typed for TNF-alpha(-308G/A), TGF-beta1(codon 10T/C, codon 25C/G), IL-10(-1082G/A, -819C/T, -592C/A), IL-6(-174C/G), and IFN-gamma(+874T/A) polymorphisms using a SSP-PCR kit. An AR episode was defined based on clinical and histological findings (Banff criteria). RESULTS.: The incidence of AR was 17%. In univariate statistical analyses recipients with TNF-alpha -308AA-genotype were found to be at a significantly increased risk for rejection (odds ratio [OR] 5.0, 95% CI 3.0-8.3, P = 0.003). The association was independent from the patient-donor HLA-mismatch status. In addition, patients with IL-10 ACCACC, ATAATA, GCCATA (-1082A/G, -819C/T, -592C/A, respectively) haplotypes were predisposed to rejection (OR 1.9, 95% CI 1.1-3.1, P = 0.016). Further, the combination of recipient TGF-beta1 25GG-genotype and donor IL-10 -819T-allele was associated with rejection (OR 1.8, 95% CI 1.1-3.0, P = 0.027). These variables remained significant risk factors also in a multivariate logistic regression analysis. The incidence of DGF was 22%. The risk was increased by a donor TNF-alpha -308GA-genotype (OR 1.6, 95% CI 1.1-2.6, P = 0.040). CONCLUSIONS: Our results confirm that cytokine gene polymorphisms influence the outcome of kidney transplantation. Our data especially identify the TNF-alpha -308AA-genotype as a factor predisposing for AR episodes.

The PredictAD project: development of novel biomarkers and analysis software for early diagnosis of the Alzheimer's disease.

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.

Feasibility of diagnosing subclinical renal allograft rejection in children by whole blood gene expression analysis.

BACKGROUND: 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. METHODS: Whole blood samples taken at the time of 3- or 6-month protocol biopsy in 31 pediatric renal transplant recipients, 13 of whom had biopsy-proven subclinical rejection (SCR), were studied. The samples were collected into tubes containing an RNA stabilization reagent enabling feasible collection during a normal ward schedule. In all patients, the gene expression of candidate genes CD154 and inducible T-cell co-stimulator (ICOS) was measured. A low-density array containing 90 immunologic-related genes were measured with real-time quantitative PCR (RT-QPCR) in 10 patients. In addition, a whole genome microarray analysis was performed in eight patients. RESULTS: Neither CD154 nor ICOS gene expression was diagnostic for SCR (median expression level 1.25 vs. 1.16 and 1.95 vs. 1.61 for CD154 and ICOS, respectively). In addition, expression levels of none of the genes on the low-density array were associated with SCR. Finally, in the microarray analysis none of the found differences between SCR and normal patients' gene expression could be validated with RT-QPCR in 17 genes. CONCLUSIONS: In our relatively small series no robust whole blood gene expression biomarker for SCR was found. Further studies are needed to determine whether small changes in expression may provide a supporting diagnostic method.