Long-Term Sheep Implantation of WIMAGINE®, a Wireless 64-Channel Electrocorticogram Recorder.

This article deals with the long-term preclinical validation of WIMAGINE® (Wireless Implantable Multi-channel Acquisition system for Generic Interface with Neurons), a 64-channel wireless implantable recorder that measures the electrical activity at the cortical surface (electrocorticography, ECoG). The WIMAGINE® implant was designed for chronic wireless neuronal signal acquisition, to be used e.g., as an intracranial Brain-Computer Interface (BCI) for severely motor-impaired patients. Due to the size and shape of WIMAGINE®, sheep appeared to be the best animal model on which to carry out long-term in vivo validation. The devices were implanted in two sheep for a follow-up period of 10 months, including idle state cortical recordings and Somato-Sensory Evoked Potential (SSEP) sessions. ECoG and SSEP demonstrated relatively stable behavior during the 10-month observation period. Information recorded from the SensoriMotor Cortex (SMC) showed an SSEP phase reversal, indicating the cortical site of the sensorimotor activity was retained after 10 months of contact. Based on weekly recordings of raw ECoG signals, the effective bandwidth was in the range of 230 Hz for both animals and remarkably stable over time, meaning preservation of the high frequency bands valuable for decoding of the brain activity using BCIs. The power spectral density (in dB/Hz), on a log scale, was of the order of 2.2, -4.5 and -18 for the frequency bands (10-40), (40-100), and (100-200) Hz, respectively. The outcome of this preclinical work is the first long-term in vivo validation of the WIMAGINE® implant, highlighting its ability to record the brain electrical activity through the dura mater and to send wireless digitized data to the external base station. Apart from local adhesion of the dura to the skull, the neurosurgeon did not face any difficulty in the implantation of the WIMAGINE® device and post-mortem analysis of the brain revealed no side effect related to the implantation. We also report on the reliability of the system; including the implantable device, the antennas module and the external base station.

WIMAGINE(®): 64-channel ECoG recording implant for human applications.

A wireless 64-channel ElectroCorticoGram (ECoG) recording implant named WIMAGINE(®) has been designed for clinical applications. This active implantable medical device is able to record ECoG on 64 electrodes with selectable gain and sampling frequency, with less than 0.7 µVRMS input referred noise in the [0.5 Hz - 300 Hz] band. It is powered remotely through an inductive link at 13.56 MHz, communicates wirelessly on the MICS band at 402-405 MHz with a custom designed base station connected to a PC and complies with the regulations applicable to class III AIMD. The design of the housing and the antenna have been optimized to ease the surgery and to take into account all the requirements of a clinical trial in particular patient safety and comfort. The main features of this WIMAGINE(®) implantable device and its architecture will be presented, as well as its performances and in vivo validations.

Autoantibodies to endostatin in patients with breast cancer: correlation to endostatin levels and clinical outcome.

Circulating autoantibodies to self-antigens overexpressed by cancer cells are common in cancer patients. As specific proteins are expressed during neoangiogenesis, a similar phenomenon might occur with particular antigens of tumour vessels. Collagen XVIII, from which endostatin is cleaved, is highly expressed in the perivascular basement membrane of tumour-associated blood vessels and autoantibodies to endostatin have been reported in cancer patients. The present study analyses the incidence of naturally occurring autoantibodies to endostatin in the sera of breast cancer patients and their relation to endostatin serum levels and patient clinical outcome. Serum samples from 36 patients with localised breast cancer and 59 patients with a fully documented history of metastatic breast cancer were used. The immunoreactivity of serum samples was tested against purified recombinant human endostatin and endostatin levels were determined by immunoassay. We could detect anti-endostatin antibodies in the sera of 66% of the patients with localised disease and 42% of the patients with metastatic disease (P=0.03). There was no correlation between the presence of antibodies to endostatin and circulating levels of endostatin. The detection of autoantibodies to endostatin was associated with better prognosis in metastatic breast cancer patients (median survival time: 20 vs 8 months, P = 0.03), as was the presence of low levels of serum endostatin (median survival time: 20 vs 9 months, P = 0.007). These results show that a natural immune reaction against endostatin can occur in breast cancer patients. This could have important therapeutic implications with regard to endostatin therapy and raises the question of a possible role of this humoral reaction against endostatin in the neoplastic process.

The bacterial nucleoside N(6)-methyldeoxyadenosine induces the differentiation of mammalian tumor cells.

Contrary to bacterial DNA, mammalian DNA contains very little if any N(6)-methyldeoxyadenosine (MDA). The possible biological effect of this nucleoside on eukaryotic cells has been studied on different tumor cell lines. Addition of MDA to C6.9 glioma cells triggers a differentiation process and the expression of the oligodendroglial marker 2',3'-cyclic nucleotide 3'phosphorylase (CNP). The biological effects of N(6)-methyldeoxyadenosine were not restricted to C6.9 glioma cells since differentiation was also observed on pheochromocytoma and teratocarcinoma cell lines and on dysembryoplastic neuroepithelial tumor cells. The precise mechanism by which MDA induces cell differentiation remains unclear, but is related to cell cycle modifications. These data point out the potential interest of N(6)-methyldeoxyadenosine as a novel antitumoral and differentiation agent. They also raise the intriguing question of the loss of adenine methylation in mammalian DNA. Furthermore, the finding that a methylated nucleoside found in bacterial DNA induces a biological process might have implications in gene therapy approaches when plasmid DNAs are injected into humans.

p53 Status and gene transfer experiments using CMV enhancer/promoter.

Comparison of transfection efficiencies between different commercial reagents or methods is a matter of major concern in the field of gene therapy. Transfection efficiencies are usually evaluated by the quantification of a reporter gene expression (i.e., luciferase or lacZ) whose expression is usually driven by the CMV promoter. However, this experimental approach does not consider the possible effects of the transfection on the activity of the promoter used to drive reporter genes expression. Using p53 null fibroblasts we show that transfection efficiency estimated by the use of pCMV-luc or pCMV-betagal plasmids may be dramatically affected by the cell p53 status. These data highlight the fact that differences in p53 levels may be one of the parameters involved in the variation of transfection efficiencies observed with different cell lines. Furthermore, they point to the fact that comparison of transfection efficiencies should distinguish differences in the efficiency of transfection from differences in the level of transcription of the transgene. Finally they suggest that the known p53 down-regulation of the CMV promoter should be considered in order to avoid the nonintentional construction of transfer vectors in which the expression of a transgene down-modulates its own promoter.

An enzymatic procedure for the purification of DNA restriction fragments without gel electrophoresis and ethidium bromide staining.

A rapid and simple enzymatic method for the purification of a DNA fragment from a restriction digest was developed. The method is based on the two features of exonuclease III activity: digestion of DNA from a 3'-OH at blunt or recessed ends and failure to initiate digestion at DNA ends with four-base 3' overhangs. Herein, we establish a method for purification of a DNA restriction fragment without any physical separation via gel electrophoresis. The elimination of the ethidium bromide staining and ultraviolet irradiation steps should increase the quality and the safety of the purified DNA, a matter of major concern in the perspective of human gene therapy. In addition, since the method described does not use the visualization of the restriction fragments or their difference in size it can be used to purify a DNA fragment from a pool of DNA fragments with the same size even when microquantities of material are available.

Antibodies to endostatin in a multifocal glioblastoma patient.

Endostatin, a C-terminal fragment of collagen XVIII is involved in the regulation of neovascularisation in solid tumours in mice. However, few data are available on the concentration of endostatin protein in patients with cancer. Paradoxical results obtained in this way prompted us to investigate an antibody to endostatin. We detected antibodies to endostatin in the serum and in the tumour brain tissue of a patient with a multifocal glioblastoma, and in the serum samples from two patients with aggressive tumours. These data suggest that endostatin overexpression by tumour tissue might induce a humoral immune response.

Bacterial DNA methylation and gene transfer efficiency.

The necessary amplification step in bacteria of any plasmid currently used in DNA immunization or gene therapy introduces modification in the nucleotide sequence of plasmid DNA used in gene transfer. These changes affect the adenine and the internal cytosine in respectively all of the GATC and CC(A/T)GG sequences. These modifications which introduce 6-methyladenine and 5-methylcytosine in plasmidic DNA are the consequence of the existence of the bacterial modification systems Dam and Dcm. In eucaryotes, the presence of 5-methylcytosine at dinucleotides -CG- is involved in silencing gene expression, but the possible consequences of the presence of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in the plasmids used in gene transfer experiments are presently unknown. Since the possibility exists to obtain plasmid DNA lacking this specific bacterial pattern of methylation by using (dam(-), dcm(-)) bacteria we performed experiments to compare in vitro and in vivo gene transfer efficiency of a pCMV-luc reporter plasmid amplified either in the JM109 (dam(+), dcm(+)) or JM110 (dam(-), dcm(-)) bacteria. Data obtained demonstrated that the presence of 6-methyladenine in GATC sequences and 5-methylcytosine in the second C of CC(A/T)GG motifs does not reduce the levels of luciferase activity detected following in vitro or in vivo gene transfer. On the contrary, gene transfer with a pCMV-luc amplified in JM109 (dam(+), dcm(+)) bacteria gives greater amounts of luciferase than the same transfection performed with a plasmid amplified in the mutated JM110 (dam(-), dcm(-)) counterpart. Therefore, these data do not suggest that the use of (dam(-), dcm(-)) bacteria to amplify plasmid DNA may increase gene transfer efficiency. However, the persistence of the use of (dam(+), dcm(+)) bacteria in order to amplify plasmid DNA raises the question of the possible biological consequences of the introduction of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in eukaryotic cells or organisms.