The comparative efficacy of theta burst stimulation or functional electrical stimulation when combined with physical therapy after stroke: a randomized controlled trial.

OBJECTIVE:: To study the long-term effectiveness of Theta Burst Stimulation (TBS) or Functional Electrical Stimulation (FES) combined with Physical therapy (PT) as compared to PT alone for improving arm functions in patients with acute stroke. DESIGN:: Single blind randomized controlled trial. SETTING:: Outpatient clinics and inpatient wards at tertiary care neurology center. SUBJECTS:: Adult patients with acute middle cerebral artery territory ischemic stroke. INTERVENTIONS:: 60 patients were randomized into three groups of 20 each: TBS+PT; FES+PT; and PT alone. TBS group received intermittent TBS of ipsilesional hemisphere and continuous TBS of contralesional hemisphere while FES group received FES of paretic limb, both for four weeks. All groups received supervised physical therapy for four weeks followed by home physiotherapy for one year. OUTCOME MEASURES:: Fugl Meyer Assessment upper limb score (FMA-UL) was primary outcome measure. Patients were evaluated at baseline and subsequently at one, three and six months and one year. RESULTS:: Compared to PT group, mean FMA-UL scores were higher in TBS and FES groups at all follow-ups ( P < 0.001). From baseline to one year, mean (SD) FMA-UL scores increased from 14.9(2.1) to 55.55(2.46) in TBS group, 15.5(1.99) to 55.85(2.46) in FES group, and 14.3(2.2) to 43.3(4.22) in PT group indicating an increase of 273%, 260%, and 203% respectively. There was no difference between FES and TBS groups. CONCLUSION:: A four-week intervention with TBS or FES combined with PT produces better long-term arm functions as compared to PT alone in patients with acute stroke.

Cytogenetic basis of acute myeloid leukemia.

The chromosomes of 12 adult patients with acute leukemia were analyzed by conventional means and by Giemsa and centromeric banding techniques. Acute myeloblastic leukemia was diagnosed in 7, acute myelomonocytic leukemia in 2, and acute undifferentiated leukemia in 3. Bone marrow was aspirated from patients when in relapse or remission, and both euploid and aneuploid cells were examined. All patients showed trisomy no. 9 and many showed additional numerical or structural changes in some or all their cells. These changes included monosomy no. 21 and/or monosomy no. 8. The proportion of trisomy no. 9 cells was 30-50% in patients in full remission and up to 100% in patients in relapse; thus trisomy no. 9 might be an important marker of leukemic cells. A mechanism was proposed to explain the induction and selection of the trisomy no. 9 karotype.

Thirteen cases of leukemia in a family.

Thirteen cases of leukemia, 12 of them acute, occurred in 3 generations of a family comprising 293 members. Individual cases could not be linked to the possession of any of a range of genetic markers. Cytogenetic studies showed no constitutional chromosome abnormalities. Preliminary results of virologic studies suggested the presence of oncornaviruses in at least 1 leukemic individual in this family. This aggregation of leukemia cases likely resulted from a genetic, probably polygenic, predisposition, in association with the activity of leukemogenic factors whose nature remains to be clearly defined.

Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia.

Hypermethylation in cancer often occurs in CpG islands that span the promoter regions of tumor suppressor genes. However, it is not clear if hypermethylation is limited to single target genes or if multiple genes are simultaneously methylated. To understand the extent of aberrant de novo methylation, we have analyzed the methylation pattern of a number of tumor-related genes in leukemia from the same cohort of patients. We used bisulfite genomic sequencing to characterize the methylation pattern of the CpG islands associated with the calcitonin, estrogen receptor, E-cadherin, p15, p16, Rb, GST-Pi, and HIC1 genes in the bone marrow from 9 normal and 20 patients with acute myeloid leukaemia (AML). All of the normal control samples were essentially unmethylated for each of the eight tumor-related genes studied. In contrast, 19 of 20 (95%) of the AML patients had an abnormal methylation pattern in at least one gene, and 15 of 20 (75%) had abnormal methylation patterns in two or more of the target genes. We conclude that there is a general deregulation of CpG island methylation in leukemia and that hypermethylation is not limited to single genes, but a number of genes are methylated concurrently. Moreover, the subset of genes that are commonly methylated in leukemia appear to be cancer type specific.

Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumors.

The retinoblastoma gene (Rb) is one of the best characterized tumor suppressor genes, and its inactivation is associated with a number of cancers. Previous studies have shown, by restriction enzyme analysis, that the promoter region of the Rb gene is methylated in a significant proportion of primary retinoblastoma tumors. We now report the first detailed methylation sequence analysis of the CpG island spanning the retinoblastoma promoter from hypermethylated retinoblastoma tumors. Our results show methylation is not confined to a specific CpG site, as detected by restriction enzyme studies, but extends to essentially all 27 CpG dinucleotides spanning the retinoblastoma CpG island, including the core promoter. The methylation pattern from each tumor DNA sample is different, ranging from densely to sparsely methylated profiles. Single CpG sites, in particular the E2F transcription factor binding site, as well as blocks of CpGs, were undermethylated in some tumor samples. Possible interference of methylation could be due to the binding of sequence-specific protein factors at these sites in the tumor cells. This study highlights that the dynamics of DNA methylation in cancer cells are clearly different from normal cells and gives an insight into the mechanism of abnormal methylation of CpG islands in cancer cells.

Bladder cancer xenografts: a model of tumor cell heterogeneity.

Twenty bladder biopsies from patients with primary transitional cell carcinoma were inoculated into nude mice. To date, eleven of these have grown as primary implants and three serially transplantable xenograft lines (UCRU-BL-12, UCRU-BL-13, UCRU-BL-14) have been established. The histological and ultrastructural features of human transitional cell carcinoma have been maintained in each line. Despite a relatively uniform histological appearance, several indices of occult tumor heterogeneity have been revealed. Immunocytochemical staining was negative for beta-subunit human chorionic gonadotrophin but positive for carcinoembryonic antigen only in areas of squamous differentiation. All three tumors bound peanut lectin. Flow cytometric DNA analysis of UCRU-BL-13 showed multiple aneuploid peaks, separate populations being demonstrated in different xenografts of the same generation. However, the morphologies of these tumors remained identical. On initial implantation UCRU-BL-12 and UCRU-BL-14 were near diploid but aneuploid populations became apparent with increasing passage number. Each xenograft line caused cachexia in the host mice. Treatment with the cisplatin analogue, isopropyl platinum, ameliorated the cachexia displayed by mice carrying UCRU-BL-14 but did not cause tumor regression. UCRU-BL-12, when tested with cisplatin, isopropyl platinum, and carboplatin, showed equivalent growth retardation with each drug. These xenografted human bladder cancers may be useful models for the study of heterogeneity of the tumor populations in bladder cancer and for the evaluation of new approaches to treatment.

Buoyant density and hybridization analysis of human DNA sequences, including three satellite DNAs.

Total human DNA was fractionated from the three types of Cs2SO4 gradient used to prepare satellites I, II and III. Three satellite DNAs were found: satellite I with a mean buoyant density of 1.6888 g/ml comprising about 1.3% of the total, satellite II with a mean buoyant of 1.696 g/ml, comprising about 1% of the total an satellite III with a mean buoyant density of 1.699 g/ml comprising about 2.2% of the total. The buoyant densities of these satellites after purification were 1.686, 1.694 and 1.697 m/gl, respectively. A preparation with the attributes of satellite IV was isolated from the shoulder region of a satellite III preparative gradient. In situ hybridization using complementary RNA showed that the three satellites were located predominantly on chromosomes 9, Y, 15 and 1. Satellite II also showed marked hybridization to chromosome 16. Satellites I and II and III cross-hybridized to each other but satellites I and II did not. On the basis of our hybridization data, we suggest that some of the same sequences which comprise satellite III also comprise satellite I an II.

Characterization of endocrine-rich monolayers of human fetal pancreas that display reduced immunogenicity.

Fibroblast-free insulin-secreting monolayers of human fetal pancreas (14-20 wk of gestation) were formed by plating isletlike cell clusters (ICCs) obtained from partially digested pancreases on plates coated with bovine corneal matrix. Human fetal pancreatic cells, freshly digested with collagen, displayed a 17-fold response to human peripheral blood lymphocytes (HPBLs) in mixed-lymphocyte culture. After 14 days in culture, monolayers derived from ICCs exhibited a smaller, twofold response to HPBLs. By comparison, in monolayers produced from single-cell suspensions, fibroblast overgrowth remained a problem. The endocrine component of the monolayers was 65 +/- 13 and 43 +/- 8%, respectively, with the number of beta-cells being 51 and 9%. Cells from both monolayers displayed increased insulin release when exposed to 10 mM theophylline, 10 mM Ca2+, and 0.6-1.3 microM 12-O-tetradecanoylphorbol-13-acetate but not to 20 mM glucose. Monolayers derived from ICCs synthesized DNA, proinsulin, and protein. This study showed that it is possible to establish an endocrine-rich monolayer of human fetal pancreas that has greatly reduced immunogenicity. The existence of residual activity to HPBLs suggests some additional form of immunosuppression is required to prevent rejection of this tissue when grafted into diabetic patients. Subculturing and cryopreservation may also be needed to achieve adequate numbers of beta-cells for clinical transplantation.

Sequence relationships of three human satellite DNAs.

The simple sequence components of three human classical satellite DNAs have been defined, and some segments of each satellite have been sequenced. Each of the classical satellites I, II and III was found to contain, as a major component, a single family of simple repeated sequences. The three simple-sequence families have been called satellites 1, 2 and 3, to indicate the enrichment of each in one of the classical satellites I, II and III, and to differentiate them from these classical satellites, which also contain other repeated components. Satellite 3, the simple sequence component of classical satellite III, when digested with the restriction endonuclease HinfI, forms a ladder based on a repeat of five base-pairs, 5' A-T-T-C-C. The HinfI ladder was shown to be composed of repeated elements with the general sequence 5' (A-T-T-C-C)n-A-TC-T-C-G-G-G-T-T-G. Satellite 2, the simple sequence component of classical satellite II, is digested by HinfI into a large number of very small fragments, of length 10 to 80 base-pairs. These were found to contain the simple repeat 5' A-T-T-C-C, in a highly diverged form. Analysis of satellite 2 sequences suggested that the five base-pair repeat was originally amplified as a higher-order repeat like that of satellite 3. However, the main tandemly repeated segments of satellite 2 in the human genome are much longer, and the simple sequence elements on which they are based are quite degenerate. Satellite 1, the simple sequence component of classical satellite I, is digested by the restriction endonuclease RsaI into a ladder of fragments less than 150 base-pairs in length. These ladder fragments were found to be formed by the loss of RsaI sites from two related A + T-rich sequences, A (17 base-pairs) and B (25 base-pairs), arranged in alternating arrays, -A-B-A-B-A-. Analysis of a large number of cloned fragments from the RsaI ladder of satellite 1 showed that the tandem arrays, -A-B-A-B-A, have a more complex arrangement, with apparent amplification of segments containing particular sequence variants of the repeat units, A and B. No sequence relationship was evident between the repeat elements of satellite 1 and those of satellites 2 and 3.

Binding of proteins from embryonic and differentiated cells to a bidirectional promoter contained within a CpG island.

We have analysed binding sites of nuclear protein factors to a CpG island (HTF9), which contains the promoter for a pair of overlapping, divergently-transcribed "housekeeping" genes. Using DNaseI protection assays with extracts from a range of differentiated and undifferentiated cell lines, including mouse embryonic stem (ES) and embryonal carcinoma (EC) cells, we located multiple protein binding sites on HTF9. Most of the sites were outside the defined core promoter and could bind to previously identified transcription factors. These included constitutive, inducible and apparently tissue-specific factors in an extremely asymmetric array relative to the transcription start sites of the two genes. A number of sites showed different binding specificities or affinities in different cell types, including ES cells. However, we found no factors that were specific for both ES and EC cells, and no protein-binding site protected exclusively in undifferentiated embryonic cells.

Cancer-specific region of hypermethylation identified within the HIC1 putative tumour suppressor gene in acute myeloid leukaemia.

Abnormal DNA methylation has been found to be a common feature in cancer cells, although the mechanism of this alteration remains poorly understood. HIC1 is a putative tumour suppressor gene on chromosome 17p13.3 and is hypermethylated in a number of cancers including leukaemia. In this study, using bisulphite genomic sequencing, we have identified a 'boundary' sequence within the HIC1 CpG island that shows a marked junction between methylated and unmethylated DNA in normal haematopoietic cells. Surprisingly, this boundary of differential methylation lies exactly between the intron 2 and exon 3 junction. In contrast to normal haematopoietic cells, hypermethylation extends past this boundary at a high frequency (83%) in newly diagnosed acute myeloid leukaemias (AML). Identification of the hypermethylated boundary sequence not only provides the first step in understanding the mechanisms that normally protect CpG islands from de novo methylation but also may prove to be a useful cancer-specific marker.

Increased DNA methyltransferase expression in leukaemia.

Aberrant DNA methylation has been observed consistently in many human tumours, in particular in the CpG islands of tumour suppressor genes, but the underlying mechanism of these changes remains unclear. To determine whether DNA methyltransferase expression is increased in leukaemia, we developed a standardised competitive RT-PCR assay to measure the level of DNA methyltransferase transcripts. Using this assay on bone marrow RNA samples from 12 patients with acute leukaemia, we observed a 4.4-fold mean increase in the level of DNA methyltransferase mRNA compared with normal bone marrow. These results support but do not prove the hypothesis that an increase in DNA methyltransferase activity is associated with malignant haematological diseases and may constitute a key step in carcinogenesis.

Fragment filtration: a method for the accurate determination of flow cytometric kinetic data from bone marrow aspirates.

The extent to which bone marrow obtained by conventional aspiration is contaminated by peripheral blood has been confirmed and quantitated. In marrow aspirates from normal subjects the median percentage of nucleated cells that had originated from the peripheral blood was 32% (range 2.5%-64%), in patients with acute leukemia 23% (range 0.5%-96.5%), in patients with chronic leukemia 59% (range 17%-76%), and in patients with lymphoma 31% (range 0.5%-74%). Flow cytometric (FCM) DNA analysis of conventional marrow aspirates from a range of subjects significantly underestimated the proportions of S-phase cells present, when compared with results from trephines obtained at the same time. Having shown, using 51Cr-labeled red cells in mice, that circulating red cells do not reenter the marrow parenchyma, a mathematical correction for contaminating blood similar to that described by Holdrinet et al. was devised. This correction improved the S-phase cell estimate from aspirated marrows, and the corrected values were not significantly different from values from paired trephine samples. A previously described technique for collecting fragments by filtration of aspirated marrow has been adapted for FCM analysis as a more direct way of overcoming problems due to blood contamination. This method was shown to yield estimates of S-phase cells not significantly different from those in paired marrow trephines and offers an alternative to routine trephine biopsies for FCM analysis of marrow cell kinetics.

Fine needle aspiration sampling of bone marrow for DNA flow cytometric analysis.

Fine needle aspiration (FNA) was used to obtain bone marrow samples from 57 patients with a variety of hematologic disorders, and from three normal subjects. We confirm that the technique is well tolerated and show that reliable cytokinetic and cell differential data can be obtained provided marrow fragments are isolated for analysis. This requires aspiration of a larger volume of marrow than originally described but this can still be achieved with minimal discomfort to the patient. The technique thus provides a means by which marrow samples could be obtained at frequent intervals in the monitoring of chemotherapy.

Cell cycle homogeneity in bone marrow samples from different sites: flow cytometric evaluation of multiple samples from sheep.

The distributions of cells in each of the phases of the cell cycle, determined by flow cytometry (FCM), were compared in multiple marrow trephine biopsy samples from 3 sites (both iliac bones and sternum) in 5 sheep. Within any one animal no significant differences could be found between the proportions of cells in the G0/G1, S or G2 + M phases of the cycle from different sites. Differences between animals were detected and these were consistent for any of the sites sampled. We conclude that the proliferative characteristics of marrow cells as determined by FCM in any one animal at one time are comparable at anatomically distinct marrow sites, and that a sample from one site is representative of the whole.

Long-term bone marrow culture induces terminal differentiation of human myeloid leukemic cells.

Bone marrow cells from a patient with acute myeloblastic leukemia were simultaneously cultured in vitro under conditions that favored the survival of either (1) leukemic progenitors (leukemic suspension culture), or (2) normal progenitors (long-term bone marrow culture). Whereas cells that were morphologically primitive and cytochemically leukemic persisted in leukemic suspension culture, they were progressively and completely replaced in long-term bone marrow culture by neutrophilic granulocytes and subsequently by macrophages. However, Auer rods were present in the maturing myeloid cells, including polymorphonuclear neutrophils, between the 7th and 30th days of long-term bone marrow culture, indicating that they were derived directly from the original leukemic population. This observation suggests that, at least in some patients, selection of cells with the potential for terminal differentiation may be the underlying mechanism responsible for the purging properties that have been attributed to long-term bone marrow culture.

A simple and rapid immunological technique for visualising chromosome-mediated gene transfer (CMGT).

A method is described for visualising chromosome-mediated gene transfer (CMGT) by detecting chromosomes labelled with bromodeoxyuridine (BrdU) using a monoclonal antibody to BrdU. In this experiment, the CCRF-CEM T cell line was grown in the presence of BrdU and the labelled chromosomes were isolated and transfected into human embryonic fibroblasts. Uptake and retention of chromosomes were compared for transfection with either PEG or DMSO treatments. Following transfection the labelled chromosomes could be visualised in recipient cells using a monoclonal antibody to BrdU, followed by immunoperoxidase staining. Chromosome uptake into cells was similar for both DMSO and PEG treatments and was a relatively frequent event; about 1 in 5 recipient cells had labelled material present. This technique can be used to assess the technical aspects of the earliest stages of chromosome-mediated gene transfer.

Pig fetal pancreatic monolayers. A model of potential use in transplantation.

Endocrine-rich monolayers of pig fetal pancreas that are free of fibroblasts have been established with the ultimate aim of providing guidelines for the culture of the human equivalent. The immunogenic potential of the monolayers--hence their capacity to be grafted--has also been analyzed. Fetuses ranging from 50 to 90 days were used, and, following digestion with collagenase (4 mg/ml, 15-20 min), the pancreatic suspension was plated onto tissue culture vessels containing RPMI 1640. The fetal calf serum concentration was kept low (5%) initially to inhibit fibroblast proliferation, but subsequently increased to 7%. Monolayers from a typical litter of 8-10 fetal pigs produced 6-8 x 10(8) viable epithelial cells by day 10 of culture, of which 75% were endocrine cells. This represents an 8-fold increase in a two-week period. The ratio of beta:alpha:delta:pancreatic polypeptide cells was 19:33:18:5. These monolayers synthesized both DNA, (pro)insulin and protein, and displayed increased insulin release when exposed to 10 mM theophylline, 10 mM Ca2+ and 1.3 microM 12-0-tetradecanoyl-phorbol-13-acetate. Static stimulation with 20 mM glucose however, did not elicit a response in insulin secretion. These cells displayed no reaction to allogeneic lymphocytes in a mixed lymphocyte culture, whereas freshly obtained porcine epithelial cells did. Methods may need to be found to increase the proportion of B cells in this enriched endocrine cell population. In general however, guidelines have been established that may be useful in developing a monolayer of human fetal pancreatic cells with the eventual aim of transplantation. The reduction in immunogenicity of the pig fetal pancreatic cells suggests that they too might be a potential source for transplantation.

In vitro studies of leukocyte labeling with technetium-99m.

A method for labeling leukocytes in vitro with 99mTc is described. Separated leukocytes are incubated with 99mTc, followed by reduction with stannous chloride and washing with acid citrate dextrose solution. Maximum labeling occurs after at least 5 min incubation with pertechnetate, followed by at least 10 min incubation with stannous chloride. Labeling is similar at room temperature and at 37 degrees C. The labeled leukocytes are viable, and reutilization of label does not occur in vitro. In acid conditions (pH 5.2), the elution of 99mTc from leukocytes is comparable with that of 32P-diisopropylfluorophosphate, but 99mTc elution is greater at pH 7.2--7.4. Neutrophils label more heavily with 99mTc than do monocytes, lymphocytes, erythrocytes, or platelets.

Drug-induced aplastic anaemia and agranulocytosis. Incidence and mechanisms.

Aplastic anaemia and agranulocytosis are uncommon but serious adverse effects of drug therapy. They result from an adverse interaction between the drug and the haemopoietic pathway in certain susceptible individuals. The nature of this idiosyncratic interaction differs for different drugs and possibly for different individuals. In some instances an immune mechanism might be implicated, in others the patient's cells might carry a genetic susceptibility to the drug, while yet other patients might metabolise the drug abnormally. The idiosyncratic nature of these effects has made their investigation difficult, but experimental studies have allowed some progress in our understanding. In a practical sense, however, responsibility for preventing these problems will remain with clinicians, who should be alert to the risks and revise their prescribing habits accordingly.