Restoring movement representation and alleviating phantom limb pain through short-term neurorehabilitation with a virtual reality system.

BACKGROUND AND OBJECTIVE: We developed a quantitative method to measure movement representations of a phantom upper limb using a bimanual circle-line coordination task (BCT). We investigated whether short-term neurorehabilitation with a virtual reality (VR) system would restore voluntary movement representations and alleviate phantom limb pain (PLP). METHODS: Eight PLP patients were enrolled. In the BCT, they repeatedly drew vertical lines using the intact hand and intended to draw circles using the phantom limb. Drawing circles mentally using the phantom limb led to the emergence of an oval transfiguration of the vertical lines ('bimanual-coupling' effect). We quantitatively measured the degree of this bimanual-coupling effect as movement representations of the phantom limb before and immediately after short-term VR neurorehabilitation. This was achieved using an 11-point numerical rating scale (NRS) for PLP intensity and the Short-Form McGill Pain Questionnaire (SF-MPQ). During VR neurorehabilitation, patients wore a head-mounted display that showed a mirror-reversed computer graphic image of an intact arm (the virtual phantom limb). By intending to move both limbs simultaneously and similarly, the patients perceived voluntary execution of movement in their phantom limb. RESULTS: Short-term VR neurorehabilitation promptly restored voluntary movement representations in the BCT and alleviated PLP (NRS: p = 0.015; 39.1 ± 28.4% relief, SF-MPQ: p = 0.015; 61.5 ± 48.5% relief). Restoration of phantom limb movement representations and reduced PLP intensity were linearly correlated (p < 0.05). CONCLUSIONS: VR rehabilitation may encourage patient's motivation and multimodal sensorimotor re-integration of a phantom limb and subsequently have a potent analgesic effect. SIGNIFICANCE: There was no objective evidence that restoring movement representation by neurorehabilitation with virtual reality alleviated phantom limb pain. This study revealed quantitatively that restoring movement representation with virtual reality rehabilitation using a bimanual coordination task correlated with alleviation of phantom limb pain.

CT angiography for surgical planning in face transplantation candidates.

SUMMARY: Facial allotransplantation replaces missing facial structures with anatomically identical tissues, providing desired functional, esthetic, and psychosocial benefits far superior to those of conventional methods. On the basis of very encouraging initial results, it is likely that more procedures will be performed in the near future. Typical candidates have extremely complex vascular anatomy due to severe injury and/or multiple prior reconstructive attempts; thus, each procedure is uniquely determined by the defects and vascular anatomy of the candidate. We detail CT angiography vascular mapping, noting the clinical relevance of the imaging, the angiosome concept and noninvasive delineation of the key vessels, and current controversies related to the vascular anastomoses.

Inhibition of AHR transcription by NF1C is affected by a single-nucleotide polymorphism, and is involved in suppression of human uterine endometrial cancer.

Involvement of the aryl hydrocarbon receptor (AHR) in carcinogenesis has been suggested in many studies. Upregulation of AHR has been reported in some cancer species, and an association between single-nucleotide polymorphisms (SNPs) of AHR and cancer risk or cancer development has also been reported. This evidence suggests the involvement of some specific SNPs in AHR transcriptional regulation in the process of carcinogenesis or cancer development, but there have been no studies to elucidate the mechanism involved. In this study, we identified the transcription factor Nuclear Factor 1-C (NF1C) as a candidate to regulate AHR transcription in a polymorphism-dependent manner. SNP rs10249788 was included in a consensus binding site for NF1C. Our results suggested that NF1C preferred the C allele to the T allele at rs10249788 for binding. Forced expression of NF1C suppressed the activity of the AHR promoter with C at rs10249788 stronger than that with T. Moreover, expression analysis of human uterine endometrial cancer (HEC) specimens showed greater upregulation of AHR and downregulation of NF1C than those of normal endometrium specimens. Sequence analysis showed HEC patients at advanced stages tended to possess T/T alleles more frequently than healthy women. We also demonstrated that NF1C suppressed proliferation, motility and invasion of HEC cells. This function was at least partially mediated by AHR. This study is the first to report that a polymorphism on the AHR regulatory region affected transcriptional regulation of the AHR gene in vitro. Because NF1C is a tumor suppressor, our new insights into AHR deregulation and its polymorphisms could reveal novel mechanisms of genetic susceptibility to cancer.

Effect of transient TCDD exposure on immortalized human trophoblast-derived cell lines.

Low level, antenatal exposure to dioxins is associated with low birth weight, which in turn is associated with long-term sequelae. We exposed the human extravillous cytotrophoblast (EVT) lines HTR-8/SV40 and TCL1 to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and assessed cell growth, invasion, and differentiation. TCDD had no effect on cell proliferation, invasion, or tube formation in Matrigel. The EVT-derived cells expressed a functional aryl hydrocarbon receptor protein; however, TCDD exposure did not alter expression levels of proteins involved in EVT differentiation in early pregnancy, including hypoxia-inducible factor 1A (HIF1A), vascular endothelial growth factor (VEGF), Integrin A1, A6, and AVB3. These results suggest that the reduction in fetal weight induced by dioxin is not the result of vascular remodeling via EVT dysfunction.

Semi-allogeneic dendritic cells injected via the intratumoural injection route show efficient antitumour effects in cooperation with host-derived professional antigen-presenting cells.

Dendritic cells (DC)-based immunotherapy is a potent anticancer modality. In DC-based immunotherapy, allogeneic DC may be an alternative source, but the usefulness of allogeneic DC in DC-based immunotherapy is still controversial. When used for immunotherapy, three factors may affect the efficiency of an allogeneic DC-driven antitumour response: (1) survival time, which is affected by T-cell alloresponses; (2) major histocompatibility complex incompatibility with the host cells in the context of antigen presentation; and (3) the role of host-derived professional antigen-presenting cells (pAPC). In addition, it is unclear which injection route is preferable when using allogeneic DC. In this study, we demonstrate that semi-allogeneic DC, which share half of the genes of the recipient, are more effective when used via the intratumoural (i.t.) injection route, rather than the subcutaneous (s.c.) injection route, for the induction of efficient antitumour effects and the generation of a significant tumour-specific CD8(+) T-cell response. The i.t. route has the advantage of not requiring ex vivo pulsation with tumour lysates or tumour antigens, because the i.t.-injected DC can engulf tumour antigens in situ. Allogeneic bone marrow transplantation (BMT) models, which permit us to separately assess the three factors described previously, show that while all three factors are important for efficient antitumour effects, the control of the alloresponse to injected DC is the most crucial for host-derived pAPC to function well when DC are administered intratumourally. This information may be useful for DC-based cancer immunotherapy under circumstances that do not allow for the use of autologous DC.

Gene expression profiles by microarray analysis during matrigel-induced tube formation in a human extravillous trophoblast cell line: comparison with endothelial cells.

Endovascular differentiation of extravillous cytotrophoblasts (EVT) during placentogenesis induces remodeling of spiral arteries that increases blood flow toward the intravillous space and is required for maintaining pregnancy. To address the molecular mechanisms involved in this differentiation, we investigated the gene expression profile during matrigel-induced tube formation in TCL1 cells, a human immortalized EVT cell line, and HUV-EC-C, human umbilical vessel endothelial cells, and compared their profiles. The numbers of genes that showed significant up-regulation (>3-fold expression at both 3 and 6h, and/or >5-fold expression at either 3 or 6h) during tube formation and significant down-regulation (0.33-fold expression at both 3 and 6h, and/or less than 0.2-fold expression at either 3 or 6h), were 969 and 659 in TCL1, respectively. In HUV-EC-C, the numbers of genes that showed significant up-regulation and down-regulation were 86 and 65, respectively. Only 73 of 1628 genes that showed significant expression changes in TCL1 were common with HUV-EC-C. The genes showing significant expression change specifically in TCL1 were associated with cellular, metabolisms, proliferation, anti-apoptosis, proteolysis adhesion, and some known to be involved in EVT differentiation or related to angiogenesis. The gene expression profile in EVT during tube formation is very different from that of endothelial cells. Further investigations based on the current data may help to elucidate mechanisms of normal and abnormal placentogenesis.

Hypoxia inducible factor 1 alpha regulates matrigel-induced endovascular differentiation under normoxia in a human extravillous trophoblast cell line.

Extravillous trophoblast (EVT) cells mimic endothelial cells during angiogenesis, inducing remodeling of the spiral arteries that increases blood flow toward the intravillous space. We have previously shown that signals involving the vascular endothelial growth factor (VEGF) axis are essential for endovascular differentiation through integrin signaling from the extracellular matrix: This was accomplished with use of the human EVT cell line TCL1, which shows tube formation that specifically recalls morphological changes in endothelial cells. To investigate endovascular differentiation in EVT further, we investigated the role of hypoxia inducible factor (HIF)1A, a subunit of HIF1 transcription factor that regulates not only adaptive responses to hypoxia, but also many cellular functions under normoxia, which was up-regulated in DNA microarray analysis during matrigel-induced endovascular differentiation under normoxia. HIF1A induces VEGF and ITGAV/ITGB3 aggregation, actions known to be important for cellular survival and endovascular differentiation in EVT. Inhibition of HIF1A up-regulation using siRNA introduction or chemical inhibition suppressed hypoxia-responsive element transcriptional activity, VEGF induction, ITGAV/ITGB3 aggregation accompanied by the inhibition of tube formation in TCL1 cells. These results suggest that HIF1A has a crucial role in regulating EVT behavior including matrigel-induced endovascular differentiation under normoxia.

Squamous cell carcinoma arising in mature cystic teratoma of the ovary: an immunohistochemical analysis of its tumorigenesis.

AIMS: Squamous cell carcinoma (SCC) is the most common form of malignant transformation in mature cystic teratoma (MCT) of the ovary. Some investigators have suggested the possibility of origin from columnar epithelium. The aim of this study was to analyse such tumours immunohistochemically to elucidate their histogenesis. METHODS AND RESULTS: The expression of cytokeratin (CK) 10 and CK18 was examined in 21 samples of SCC arising in MCT. The expression of CK10 and CK18 was also assessed in SCCs arising in different organs (skin, vulva, lung and uterine cervix) for the purpose of comparison. SCC in MCT expressed CK10 in 7/21 cases [33.3%, 95% confidence interval (CI) 0.12-0.53] and CK18 in 14/21 cases (66.7%, 95% CI 0.46-0.87). SCC in MCT expressed CK10 less frequently, but CK18 more frequently, as is the case in SCCs of the uterine cervix (CK10, 20%; CK18, 70%) and the lung (CK10, 5%; CK18, 90%), both of which are derived from columnar epithelium by squamous metaplasia. CONCLUSIONS: SCC in MCT may be derived from metaplastic squamous epithelium.

Spontaneous rupture of sacrococcygeal teratoma associated with acute fetal anemia.

Sacrococcygeal teratoma (SCT) is the most common congenital tumor, with affected fetuses having a high risk of perinatal complications and death. We report a case of a fetus with an SCT that developed acute anemia due to spontaneous rupture of the tumor in utero. The fetus was referred at 25 weeks' gestation for evaluation of a large solid and cystic mass in the sacral region. There were no signs of hydrops or placentomegaly. At 33 weeks' gestation, loss of variability in the fetal heart rate pattern was recorded. Doppler ultrasonography showed increased middle cerebral artery peak systolic velocity, suggesting fetal anemia. Markedly bloody amniotic fluid, with 82% hemoglobin F, was observed on amniocentesis, suggesting rupture of the SCT with active fetal bleeding. An emergency Cesarean section was performed. At delivery, the tumor was bleeding actively and the neonatal hemoglobin concentration was 3.1 g/dL. There were no findings of hemorrhage or necrosis within the tumor. The neonate received a blood transfusion, and surgical resection of the tumor was carried out on the first day after delivery. Postoperatively, the baby did well. We suggest that fetal SCTs run the risk of inducing acute fetal anemia due to intrauterine hemorrhage of the tumor, a finding which could lead to an earlier and more appropriate management of this condition.

Establishment of the primary imprint of the HYMAI/PLAGL1 imprint control region during oogenesis.

Imprinting within domains occurs through epigenetic alterations to imprinting centers (ICs) that result in the establishment of parental-specific differences in gene expression. One candidate IC lies within the imprinted domain on human chromosome region 6q24. This domain contains two paternally expressed genes, the zinc finger protein gene PLAGL1 (ZAC/LOT1) and an untranslated mRNAcalled HYMAI. The putative IC overlaps exon 1 of HYMAI and is differentially methylated in somatic tissues. In humans, loss of methylation within this region is seen in some patients with transient neonatal diabetes mellitus, and hypermethylation of this region is found in ovarian cancer and is associated with changes in expression of PLAGL1, suggesting that it plays a key role in regulating gene expression. Differential methylation within this region is conserved in the homologous region on mouse chromosome 10A and is present on the maternal allele. In this paper, we report that DNA methylation is established during the growth phase of oogenesis and that this coincides with the establishment of monoallelic expression from this region lending further support to the hypothesis that this region functions as an IC.

Expression of DCC and netrin-1 in normal human endometrium and its implication in endometrial carcinogenesis.

OBJECTIVE: Although DCC has been considered as a candidate tumor suppressor, the roles it plays in the uterine endometrium and in the carcinogenic process remains unclear. To define these roles more clearly, we examined the expression of DCC and its ligand, netrin-1, in the normal endometrium and in endometrial cancer. METHODS: The expression of DCC and netrin-1 in normal endometrial glands and in cancer cell lines was examined by RT-PCR and immunohistochemistry. The effects of exogenous DCC and netrin-1 expression were observed together with the respective expression vector transfection. RESULTS: Endometrial glands in the proliferative and early secretory phase expressed both DCC and netrin-1, but glands in the late-secretory phase tended to silence DCC expression. In addition, all of the endometrial cancer cell lines lost normal DCC expression. Restored DCC expression in the cancer cell lines in the absence of netrin-1 induced apoptosis. However, no changes were observed in the presence of netrin-1. CONCLUSION: Our observations suggest that DCC/netrin-1 signaling may commit cells to the transition of endometrial gland architecture or function from a proliferating to a secretory phase. In addition, the silencing of DCC expression may contribute to the escape of endometrial cancer cells from a DCC-regulated apoptotic program, thereby promoting malignant phenotypes.

Growth-associated gene expression profiles by microarray analysis of trophoblast of molar pregnancies and normal villi.

We used microarray analysis to investigate expression profiles of 589 known genes committed to cell growth control to characterize regulatory circuitry for cell proliferation in complete moles (CMs). CMs are characterized by hyperplastic trophoblast and have a high propensity to give rise to choriocarcinoma. Characteristic alterations in gene expression profiles were observed when compared with normal villi. Fifty-seven genes were significantly up-regulated in CMs and involved the Ras-Map kinase 3, Jak-STAT5, and Wnt signal pathways, implicating growth factor or cytokine-mediated signal pathways in the trophoblastic hyperplasia of CMs. Several genes associated with anti-apoptosis, cell structuring, and/or cell attachment were also up-regulated in CMs. In contrast, relatively fewer genes were down-regulated and these involved IGFBPs, versican, interleukin-1, tumor necrosis factor receptor, CD44, and RAD52. Genes identified in this study may elucidate regulation mechanisms of trophoblastic proliferation and mechanisms causing a pathological phenotype in CMs.

Sodium butyrate induces growth arrest and senescence-like phenotypes in gynecologic cancer cells.

We demonstrated here the growth-suppressing effects of sodium butyrate (NaB) on human endometrial and ovarian cancer cells. The arrest of cells at the G1 checkpoint accounted for this effect. NaB-mediated p21 might arrest endometrial and ovarian cancer cells at the G0/G1 phase by eliciting pRb unphosphorylation. To demonstrate the role of pRb regulation by p21, we measured the sensitivity to NaB of cervical cancer cells in which pRb had been inactivated by HPV E7. The cervical cancer cells displayed a sensitivity in NaB-mediated G2/M arrest in addition to their sensitivity in G0/G1 arrest. Arrest at G0/G1 and G2/M accompanied induction of senescence-like phenotypes (SLPs). Most importantly, the effect of NaB on senescence induction was not coupled with the predominance of hypophosphorylated pRb forms in the cervical cancer cells. This suggested that NaB had the potential to elicit SLPs through p21-mediated withdrawal from cell cycle progression. The consequences of p21 induction were manifold. The effects of NaB on gynecologic cancer cell growth indicated its potential use in cancer treatment. NaB was effective even in the cancer cells with mutant p53 and/or Rb genes by eliciting cell senescence.

A conserved imprinting control region at the HYMAI/ZAC domain is implicated in transient neonatal diabetes mellitus.

Transient neonatal diabetes mellitus (TNDM) is associated with intra-uterine growth retardation, dehydration and a lack of insulin. Some TNDM patients exhibit paternal uniparental disomy (UPD) of chromosome 6q24, where at least two imprinted genes, HYMAI and ZAC, have so far been characterized. Here we show that the differentially methylated CpG island that partially overlaps mZac1 and mHymai at the syntenic mouse locus is a likely imprinting control region (ICR) for the approximately 120--200 kb domain. The region is unmethylated in sperm but probably methylated in oocytes, a difference that persists between parental alleles throughout pre- and post-implantation development. We also show that within this ICR, there is a region that exhibits a high degree of homology between mouse and human. Using a reporter expression assay, we demonstrate that this conserved region acts as a strong transcriptional repressor when methylated. Finally, we provide in vivo evidence that in the majority of TNDM patients with a normal karyotype, there is a loss of methylation within the highly homologous region. We propose that this ICR regulates expression of imprinted genes within the domain; epigenetic or genetic mutations of this region probably result in TNDM, possibly by affecting expression of ZAC in the pancreas and/or the pituitary.

Genetic origin of malignant trophoblastic neoplasms analyzed by sequence tag site polymorphic markers.

OBJECTIVE: To study the causative conception of malignant gestational trophoblastic neoplasms (GTNs), we analyzed malignant GTNs by microsatellite PCR markers. METHOD: DNAs extracted from 12 malignant GTNs were subjected to PCR for five different chromosomal locations. RESULT: Of the 7 cases after a complete mole (CM), 5 were derived from androgenesis, but the remaining 2 were from normal fertilization. Of the 5 cases after nonmolar pregnancies, 2 placental site trophoblastic tumors had alleles from both parents. Of the other 3 choriocarcinomas, 1 was from normal fertilization after spontaneous abortion but 2 originated from androgenesis, suggesting that 1 was from a CM prior to the antecedent abortion, transforming after a long interval. CONCLUSION: By combining the previous cases with these, our analysis of 39 cases demonstrated that trophoblastic neoplasms can arise from at least three different modes of origin (androgenesis, normal fertilization, and parthenogenesis), and antecedent pregnancy is not always identical to the causative conception. Placental site trophoblastic tumors might have different machinery for carcinogenesis because of the predominance of paternal and maternal contributions. In addition, a long dormancy of trophoblasts before malignant transformation, especially for those originating from normal fertilization, was also suggested.

Photodynamic antisense regulation of human cervical carcinoma cell growth using psoralen-conjugated oligo(nucleoside phosphorothioate).

The antisense strategy has been applied to regulate gene expression in a sequence specific manner, which enables suppression of the proliferation of cancer cells and exploration of the functions of unknown genes. In order to generalize and to enhance the ability of the strategy, functionalization of antisense DNAs was done using a photo-crosslinking reagent, 4,5',8-trimethylpsoralen, and the possibility of photodynamic antisense regulation of gene expression was examined. Psoralen-conjugated oligo(nucleoside phosphorothioate)s (Ps-S-oligo) were prepared and used to inhibit the proliferation of human cervical carcinoma cells. Upon UVA irradiation of Ps-S-oligo treated cells, Ps-S-oligo complementary to the initiation codon region (Ps-P-As) of HPV18-E6*-mRNA of human cervical carcinoma cells inhibited drastically the cell growth (IC(50)=16 nM). In contrast, Ps-S-oligo with mismatched sequences and scrambled one showed lesser inhibitory effects than Ps-P-As. These results showed that the inhibition by Ps-S-oligo was dependent on (a) sequence, (b) UVA irradiation, (c) concentration and (d) cell line. The amount of intact HPV18-E6*-mRNA was decreased in a sequence dependent manner, indicating that the antiproliferative effect of Ps-P-As was an antisense manner. The psoralen-conjugated antisense DNA has significant potential to regulate gene expression, which may provide useful information to explore the novel gene regulating reagents.

Analysis of specific gene mutations in the transforming growth factor-beta signal transduction pathway in human ovarian cancer.

Several proteins, including transforming growth factor beta (TGF-beta) receptor type I (RI), TGF-beta receptor type II (RII), Smad2, Smad3, and Smad4/DPC4, have been identified in the transduction pathway of the tumor suppressor TGF-beta. Mutations in TGF-beta RI, TGF-beta RII, Smad2, and Smad4/DPC4 genes are associated with several human cancers. The present study examines these gene mutations in 32 human ovarian cancers and 14 patient-matched normal tissues. For the first time, mutations in the Smad2 and Smad4 genes were analyzed in relation to human ovarian cancer. Gene mutations of TGF-beta RI, TGF-beta RII, Smad2, and Smad4 were analyzed using specific primers by PCR-single-strand conformational polymorphism (SSCP), and the results revealed a frameshift mutation at codons 276-277 (CTCTGG-->CTGCGTGG) in exon 5 of TGF-beta RI in 10 of 32 tumor samples (31.3%). This mutation was associated with reduced or absent expression of TGF-beta RI protein and p53 protein in tumor tissues. We detected SSCP variants of TGF-beta RII in exon 2 in 20 of 32 tumors. Sequence analysis of these variants revealed an A to G transition at the seventh band of intron 2. In this A to G polymorphism in intron 2, 12 samples (37.5%) had A/A alleles, 12 (37.5%) had A/G alleles, and 8 (25%) had G/G alleles. We detected Smad2 SSCP variants in exon 4 in 12 of 32 tumors (37.5%). Sequence analysis revealed a 2-bp deletion in the polypyrimidine tract of intron 3, which is located at position -39 to -56 in the splice acceptor site of the intron 3-exon 4 junction. No SSCP variants were detected in the Smad4 gene. These findings suggest that mutations in the TGF-beta RI and in its signal transduction pathway are likely responsible for human ovarian carcinogenesis.

Mutation analysis of the hMTH1 gene in sporadic human ovarian cancer.

8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) is a major oxidation product in the nucleotide pool of the cell and is a potent mutagen, because it can be incorporated into DNA with equal frequency opposite either template C or A. The human MTH1 gene (hMTH1) is a homologue of the E. coli mutator gene mutT, which encodes 8-oxo-dGTPase. hMTH1 protein reduces spontaneous mutations by removing 8-oxo-dGTP from the triphosphate pool. To determine whether this gene is associated with carcinogenesis of human ovarian cancer, the present study examined, for the first time, the hMTH1 sequence in 49 ovarian cancers and 9 ovarian cancer cell lines by means of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analyses. A Gright curved arrow A transition at codon 83 was detected in one patient and one cell line (3.4%), followed by an amino acid change (valineright curved arrow methionine) which was known to cause the protein to be less active in vitro. This one base substitution was found in normal and corresponding tumor DNA, and its allele type was heterozygous. The same change has been detected in HNPCC (hereditary non-polyposis colorectal cancer) and gastric cancer patients, and thus it may not represent a mutation specific for ovarian cancer. A silent Tright curved arrow C transition at codon 119 was detected in 12 patients and 2 cell lines (24.1%). No specific mutations in hMTH1 were found in either ovarian cancer patients or cell lines. Thus, it appears that hMTH1 is not directly associated with ovarian cancer.