Functional MRI and motor behavioral changes obtained with constraint-induced movement therapy in chronic stroke.

BACKGROUND: The clinical benefits of intensive stroke rehabilitation vary individually. We used multimodal functional imaging to assess the relationship of clinical gain and imaging changes in patients with chronic stroke whose voluntary motor control improved after constraint-induced movement therapy (CIMT). METHODS: Eleven patients (37.6 ± 36.8 months from stroke) were studied by functional MRI (fMRI), transcranial magnetic stimulation (TMS), and behavioral assessment of hand motor control (Wolf Motor Function Test) before and after 2 weeks of CIMT. Individual and group-level changes in imaging and behavioral parameters were investigated. RESULTS: Increase in fMRI activation in the sensorimotor areas was greater amongst those subjects who had poor hand motor behavior before therapy and/or whose motor behavior improved notably because of therapy than amongst subjects with relatively good motor behavior already before therapy. The magnitude of CIMT-induced changes in task-related fMRI activation differed between lesioned and non-lesioned hemispheres, and the fMRI laterality index was different for paretic and non-paretic hand tasks. The corticospinal conduction time in TMS was significantly decreased after CIM therapy. CONCLUSIONS: Alterations in sensorimotor cortical activations (fMRI) and corticospinal conductivity (TMS) were observed after intensive rehabilitation in patients with chronic stroke. Activation and functional changes in fMRI and TMS correlated significantly with the degree of clinical improvement in hand motor behavior. The present data advance the understanding of the functional underpinnings of motor recovery, which may be obtained even years after the stroke.

Generation of lentivirus vectors using recombinant baculoviruses.

In spite of advances in conventional four-plasmid transient transfection methods and development of inducible stable production cell lines, production of replication-defective lentiviral vectors in clinical scale has been challenging. Baculovirus technology offers an alternative to scalable virus production as a result of fast and easy production of baculoviruses, efficient transduction of mammalian cells and safety of the baculoviruses. As a first step toward scalable lentiviral production system, we have constructed four recombinant baculoviruses: the BAC-transfer virus expresses green fluorescent protein (GFP) as a transgene and BAC-gag-pol, BAC-vesicular stomatitis virus glycoprotein G and BAC-rev express all elements required for a safe lentivirus vector generation. Following 293T cell transduction with recombinant baculoviruses functional lentiviruses were produced. Different baculovirus concentrations, mediums and transduction times were used to find optimal conditions for lentivirus production. The unconcentrated lentiviral titers in cell culture mediums were on average 2.5 x 10(6) TU ml(-1), which are comparable to titers of the lentiviruses produced by conventional four-plasmid methods. Lentiviruses produced by baculovirus method transduced HeLa cells and showed sustained GFP expression. No evidence of the formation of replication competent lentiviruses was detected by p24 enzyme-linked immunosorbent assay. Our results show that baculoviruses are an attractive alternative for the production of lentiviruses in mammalian cells.

Apolipoprotein E epsilon 4 allele is associated with increased atrophy in progressive mild cognitive impairment: a voxel-based morphometric study.

BACKGROUND: The apolipoprotein E (APOE) epsilon4 allele is a risk factor for Alzheimer's disease. Earlier studies have shown differences in brain structure according to the APOE epsilon4 status. OBJECTIVE: To assess possible differences in brain structure according to the APOE epsilon4 status in mild cognitive impairment (MCI) subjects in relation to conversion to dementia. METHODS: In a follow-up study of 56 MCI subjects, 13 MCI subjects progressed to dementia (PMCI) during a mean follow-up time of 31 months. Brain structure differences in both stable MCI (SMCI) and PMCI epsilon4 carriers and noncarriers in the baseline MRI scan were assessed with voxel-based morphometry. RESULTS: The SMCI epsilon4 carriers had atrophy in the amygdala and hippocampus compared to the SMCI noncarriers. The PMCI epsilon4 carriers revealed atrophy of the left inferior frontal gyrus and parietal cortex compared to the PMCI noncarriers. CONCLUSION: The rate of brain atrophy in certain brain areas may be increased in epsilon4-positive MCI subjects progressing to dementia.

Differentiation of subpopulations of human and murine hemopoietic stem cells by hypotonic lysis.

Both human and mouse bone marrow contain subpopulations of hemopoietic stem cells that greatly vary in their resistance to water exposure: The cells forming erythroid colonies or bursts in methyl cellulose in vitro are most sensitive to hypotonic conditions and are destroyed within 60 s in the hypotonic milieu. The murine pluripotent stem cells assayed by the spleen colony technique, as well as both murine and human myeloid stem cells assayed by the plasma clot diffusion chamber technique, displayed intermediate sensitivity and were nearly completely eliminated by 120 s of exposure to water. Both human and mouse bone marrow stem cells producing myeloid colonies in agar are most resistant to hypotonic conditions. The addition of monocyte-macrophages and lymphoid cells to water-exposed mouse bone marrow cell populations to compensate for losses did not restore either erythroid or myeloid colony formation.

Distinction of human T-cell line (HUT-102)-derived activity stimulating granulocytic colony formation in diffusion chambers in vivo from activities stimulating erythroid and mixed-colony formation in vitro.

Medium conditioned in the presence of human HUT-102 T-cell line cells contains activities stimulating human mixed (colony-forming unit, erythroid, granulocyte, macrophage, megakaryocyte) and erythroid (burst-forming unit, erythroid) colony formation in methylcellulose in vitro and granulocyte colony formation in diffusion chambers in mice. The stimulatory effect of HUT-102-conditioned medium on colony-forming unit, granulocyte diffusion chamber was also observed in diffusion chambers implanted in nude mice. The hemopoietic activities were heat stable and could be detected from serum-free conditioned medium. Chromatographically, it was possible to separate colony-forming unit, granulocyte diffusion chamber-stimulating activity from activities stimulating burst-forming unit, erythroid and colony-forming unit, erythroid, granulocyte, macrophage, megakaryocyte. On the other hand, the latter two activities were indistinguishable by the methodology used in this study. Failure to abolish the hemopoietic activities by boiling or by human T-lymphotropic retrovirus type 1 antibody indicates that human T-lymphotropic retrovirus type 1 or its components potentially present in the conditioned medium were not responsible for the stimulatory effects.

Effect of glucan, a macrophage activator, on murine hemopoietic cell proliferation in diffusion chambers in mice.

Pretreatment of mice with glucan, a potent macrophage activator, resulted in enhanced myeloid cluster and colony formation by bone marrow cells in diffusion chambers implanted into the peritoneal cavity. Simultaneously, erythroid colony formation was also augmented. In some experiments the plasma clots formed inside the chambers were dissolved, and the number of hematopoietic cells was determined. An increased yield of early proliferative cells, granulocytes, and macrophages was found in glucan-treated hosts. Concomitantly, higher leukocyte counts were noted in the peripheral blood of treated animals. These results suggest that glucan has a strong stimulatory effect on hematopoiesis. This stimulation is probably mediated by humoral factors of host animal origin rather than by direct interaction with proliferating hematopoietic precursors enclosed within the chambers.

Reduced expression of proapoptotic gene BAX is associated with poor response rates to combination chemotherapy and shorter survival in women with metastatic breast adenocarcinoma.

Bax is a homologue of Bcl-2 that promotes apoptosis. Bax protein levels were assessed by immunohistochemical methods in primary tumors derived from 119 women with metastatic breast cancer. These patients had received combination chemotherapy either with a once a month dosage schedule or in 4 weekly divided doses. The BAX immunostaining results were retrospectively compared with overall survival, time to tumor progression (TTP), and response, as well as several laboratory markers. Normal breast epithelium and in situ carcinomas immunostained positively for Bax. Marked reductions in Bax immunostaining were observed in 40 (34%) of 119 evaluable tumors. Reduced Bax correlated with shorter overall survival (median, 8.1 versus 15.7 months; P = 0.04), faster TTP (median, 2.0 versus 6.3 months; P = 0.009), and failure to respond (complete response, partial responses; 6% versus 42%, P = 0.01) in the subgroup of patients who received divided dose therapy. Reduced Bax immunostaining was not significant in the monthly dose group. When the two groups were combined, however, reduced Bax was significantly correlated in univariate analysis with failure to respond (21 versus 43% achieving complete response or partial response; P = 0.02), faster TTP (median, 3.7 versus 9.0 months; P = 0.02), and shorter survival (median, 10.7 versus 17.1 months; P = 0.04). Bax immunostaining was not significantly correlated with tumor histology, S-phase fraction, aneuploidy, p53 HER2, or cathepsin D, but was positively associated with Bcl-2 (P = 0.005). In multivariate analysis (Bax, tumor grade, and treatment group), reduced Bax was strongly associated with faster TTP (P approximately equal to 0.009) and shorter survival (P approximately equal to 0.001). Although highly preliminary, the finding suggest that loss of Bax immunostaining represents a novel prognostic indicator of poor response to chemotherapy and shorter survival in women with metastatic breast cancer, and raise the possibility that the subgroup of women with Bax-negative tumors may benefit from more aggressive therapy.

Plasma thromboplastin antecedent (Factor XI) deficiency in a black family.

A black man with a prolonged partial thromboplastin time has a severe deficiency of plasma thromboplastin antecedent (PTA) (factor XI) measured both in clotting assays and immunoassays, suggesting a diagnosis of homozygous PTA deficiency. His offspring seemed to be heterozygous carriers of PTA deficiency. Additionally, the proband and two of his children had decreased Hageman factor (factor XII) levels consistent with those of heterozygous carriers of Hageman trait. To our knowledge, this is the first case known of PTA deficiency in a black person. Its pattern of inheritance was independent of that of factor VII deficiency.

Effect of lithium on stem cell and stromal cell proliferation in vitro.

Lithium is recognized as a potent stimulator of hematopoiesis both in vivo and in vitro. Previous work has suggested that this stimulation is mediated as an indirect, humoral effect by the action of lithium upon the stromal cell population. In the present study, the effects of lithium on the stromal population were investigated using a long-term liquid marrow culture model. These findings indicate that exposure of in vitro cultures to lithium results in an increase in the total cellularity and in the number of various hematopoietic progenitor cells residing within the stromal layer. A distinct morphologically recognizable cell has not been identified as the target cell responsible for the indirect stimulation of hematopoiesis by lithium. However, two candidate radioresistant stromal cells believed to be active in the production of humoral mediators of hematopoiesis did proliferate in response to lithium exposure.

Relationship between cells forming colonies in diffusion chambers in vivo (CFU-D) and cells with high proliferative potential in vitro (HPP-CFC-1 and -2).

In vivo diffusion chambers implanted in normal mice after 5 days of bone marrow cell culture contained precursor cells that in the presence of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), interleukin 3 (IL-3), or colony-stimulating factor 1 (CSF-1), alone or in combination, formed both small and large (high proliferative potential colony-forming cells, HPP-CFC) macrophage-containing colonies in vitro. Synergistic factor from serum-free 5637 cell-conditioned medium (SF5637) enhanced HPP-CFC colony growth only in cultures containing CSF-1. Higher numbers of CSF-1- plus IL-3-responsive colony-forming cells (HPP-CFC-2) were detected in diffusion chamber colony-forming unit (CFU-D) colonies than in intercolony areas, suggesting that they were derived from cells that give rise to the diffusion chamber colony. Further study demonstrated that CFU-D colonies contained cells that formed large macrophage-containing colonies (HPP-CFC-1) in CSF-1- plus SF5637-containing cultures. These findings suggest that single cells (CFU-D) forming colonies in diffusion chambers in mice can give rise to both HPP-CFC-1 and to cells probably representing their progeny, HPP-CFC-2.

Cryopreservation of marrow and stromal progenitor cells: use of long-term liquid culture as a measure of the recovery of renewable hematopoietic and stromal cells.

Long-term liquid culture was used to assess the broad functional integrity of murine bone marrow after cryopreservation. Cryopreserved and thawed marrow contained not only renewable hematopoietic stem cells, but also stromal progenitor cells capable of transferring the hematopoietic microenvironment to liquid culture. Long-term liquid culture may be a superior method of assessing the functional potential of cryopreserved marrow when compared to stem cell assays alone.

Effect of cryopreservation on recovery of cells forming colonies in diffusion chambers in mice (CFUD).

The effect of cryopreservation on human and murine hematopoietic precursor cell survival was assessed. A large proportion of CFUC and CFUS was recovered following freezing and thawing. At the same time there was a substantial decrease in the number of cells which form myeloid (CFUDG) and megakaryocytic (CFUDM) colonies in diffusion chambers in mice. We suggest that the CFUDG and CFUDM number should be determined in the autologous bone marrow transplantation setting.

Progenitor cell numbers (CFU-GM, CFU-D, and CFU-Mix) and hemopoietic recovery following autologous marrow transplantation.

Forty-one consecutive patients were treated with high-dose chemotherapy with or without total body irradiation followed by autologous marrow transplantation. Four treatment regimens of varying intensity were used. Every patient's harvested marrow was evaluable for nucleated cell and progenitor cell loss during the cell separation and cryopreservation process. Of the 41 patients, 38 were evaluable for peripheral blood count recovery. Multivariate analysis of colony-forming cell assays and recovery of neutrophils and platelets showed a significant association with absolute numbers of post-thaw mixed colony-forming units (CFU-Mix) infused (p less than 0.002). Prefreeze CFU-Mix also correlated with recovery to a lesser degree, as did absolute numbers of nucleated cells. The number of diffusion chamber colony-forming units (CFU-D) prefreeze, but not post-thaw infused into the patient, was associated with recovery of neutrophils (p = 0.0001), but not platelets. When the precursor cell numbers were adjusted for body weight, post-thaw CFU-Mix showed the best correlation with recovery of both platelets and neutrophils. Prefreeze CFU-D per kg was also associated with recovery of neutrophils (p = 0.02). To some extent nucleated cells per kg predicted for recovery with neutrophils and platelets (p less than 0.05). When analyzed according to treatment regimen, cyclophosphamide-BCNU-VP16 (CBV) or cyclophosphamide-total body irradiation (CY/TBI) was associated with prolonged recovery compared to cyclophosphamide-adriamycin-vinblastine (CAV) or etoposide-cyclophosphamide (EC). In this setting only CFU-D number predicted neutrophil recovery (p less than 0.002). We conclude that determination of the number of total nucleated cells, CFU-D, and CFU-Mix, before cryopreservation of the sample is important in predicting hemopoietic reconstitution in autologous bone marrow transplantation.

The North Karelia Youth Programs.

Two family-based and two community- and school-based studies have been done in the province of North Karelia in eastern Finland. In the two family-based studies it was possible to decrease serum cholesterol level among children by 15%, showing that to a great extent the extremely high blood cholesterol level among Finnish children can be explained mainly by the typical Finnish diet high in saturated fat. In the first North Karelia Youth Project it was possible to affect the onset of smoking among adolescents. Two years after the program, smoking was about 30% less common in the intervention school than in the reference schools. In the eighth-year follow up survey the effect of the teacher-delivered program was still seen. In the second North Karelia Youth Project it was possible to delay the onset of smoking during the seventh grade but no permanent effect was seen. Diet was changed more among the program school students than in the reference school. These studies, done during the last 10 years, show the cardiovascular risk factors can be reduced among children and adolescents. The next main question is how to use these findings in the normal school and health care system. We have started the National Healthy School Program in Finland to assess the extent to which these programs can be implemented on the national level.

Cerebral cortex and sub-cortex lateralization in cardiovascular regulation: correlations of BOLD fMRI and heart rate variability.

The role of cerebral cortex in cardiovascular regulation has not yet been mapped in detail. Especially the lateralization of different regions that are connected to cardiovascular modulation is still unknown. In this study we used simultaneously measured electrocardiography (ECG) and blood oxygen level dependent (BOLD) fMRI to examine the correlation of cerebral cortex and sub-cortex activation and heart rate variability parameters. Correlations were calculated for 11 subjects. Regions of interest (ROIs) were predefined from observations made in previous studies. Lateralization was studied by forming ratios of left and right hemisphere activations in ROIs and calculating correlations of these to heart rate variability (HRV) parameters. Statistically significant correlations were found in every ROI.

Sensorimotor, visual, and auditory cortical atrophy in Unverricht-Lundborg disease mapped with cortical thickness analysis.

BACKGROUND AND PURPOSE: EPM1, caused by mutations in the CSTB gene, is the most common form of PME. The most incapacitating symptom of EPM1 is action-activated and stimulus-sensitive myoclonus. The clinical severity of the disease varies considerably among patients, but so far, no correlations have been observed between quantitative structural changes in the brain and clinical parameters such as duration of the disease, age at onset, or myoclonus severity. The aim of this study was to evaluate possible changes in CTH of patients with EPM1 compared with healthy controls and to correlate those changes with clinical parameters. MATERIALS AND METHODS: Fifty-three genetically verified patients with EPM1 and 70 healthy volunteers matched for age and sex underwent 1.5T MR imaging. T1-weighted 3D images were analyzed with CTH analysis to detect alterations. The patients were clinically evaluated for myoclonus severity by using the UMRS. Higher UMRS scores indicate more severe myoclonus. RESULTS: CTH analysis revealed significant thinning of the sensorimotor and visual and auditory cortices of patients with EPM1 compared with healthy controls. CTH was reduced with increasing age in both groups, but in patients, the changes were confined specifically to the aforementioned areas, while in controls, the changes were more diffuse. Duration of the disease and the severity of myoclonus correlated negatively with CTH. CONCLUSIONS: Cortical thinning in the sensorimotor areas in EPM1 correlated significantly with the degree of the severity of the myoclonus and is most likely related to the widespread stimulus sensitivity in EPM1.

Atrophic enlargement of CSF volume after subarachnoid hemorrhage: correlation with neuropsychological outcome.

BACKGROUND AND PURPOSE: Ventricular dilation and sulcal enlargement are common sequelae after aSAH. Our aim was to quantify the late ventricular dilation and volumes of the CSF spaces after aSAH and to determine if they correlate with neurologic and cognitive impairments frequently detected in these patients. MATERIALS AND METHODS: 3D T1-weighted images needed for volumetry were available in 76 patients 1 year after aSAH, along with 75 neuropsychological assessments. Volumes of CSF segments and ICV were quantified by SPM in 76 patients and 30 control subjects to determine CSF/ICV ratios. The mCMI was calculated to roughly evaluate the ventricular dilation. The contributing factors for enlarged ventricles and CSF volumes were reviewed from radiologic, clinical, and neuropsychological perspectives. RESULTS: The mCMI was higher in patients with aSAH (0.23 +/- 0.06) compared with control subjects (0.20 +/- 0.04; P = .020). In line with these planimetric measurements, the SPM-based CSF/ICV ratios were higher in patients with aSAH (35.58 +/- 7.0) than in control subjects (30.36 +/- 6.25; P = .001). Preoperative hydrocephalus, higher HH and Fisher grades, and focal parenchymal lesions on brain MR imaging, but not the treatment technique, were associated with ventricular enlargement. The clinical outcome and presence of neuropsychological deficits correlated significantly with CSF enlargement. CONCLUSIONS: Ventricular and sulcal enlargement, together with reduced GM volumes, after aSAH may indicate general atrophy rather than hydrocephalus. Enlarged CSF spaces correlate with cognitive deficits after aSAH. A simple measure, mCMI proved to be a feasible tool to assess the diffuse atrophic brain damage after aSAH.