In vivo effects of L1 coating on inflammation and neuronal health at the electrode-tissue interface in rat spinal cord and dorsal root ganglion.

The spinal cord (SC) and dorsal root ganglion (DRG) are target implantation regions for neural prosthetics, but the tissue-electrode interface in these regions is not well-studied. To improve understanding of these locations, the tissue reactions around implanted electrodes were characterized. L1, an adhesion molecule shown to maintain neuronal density and reduce gliosis in brain tissue, was then evaluated in SC and DRG implants. Following L1 immobilization onto neural electrodes, the bioactivities of the coatings were verified in vitro using neuron, astrocyte and microglia cultures. Non-modified and L1-coated electrodes were implanted into adult rats for 1 or 4 weeks. Hematoxylin and eosin staining along with cell-type specific antibodies were used to characterize the tissue response. In the SC and DRG, cells aggregated at the electrode-tissue interface. Microglia staining was more intense around the implant site and decreased with distance from the interface. Neurofilament staining in both locations decreased or was absent around the implant, compared with surrounding tissue. With L1, neurofilament staining was significantly increased while neuronal cell death decreased. These results indicate that L1-modified electrodes may result in an improved chronic neural interface and will be evaluated in recording and stimulation studies.

A computational model for estimating recruitment of primary afferent fibers by intraneural stimulation in the dorsal root ganglia.

Primary afferent microstimulation has been proposed as a method for activating cutaneous and muscle afferent fibers to restore tactile and proprioceptive feedback after limb loss or peripheral neuropathy. Large populations of primary afferent fibers can be accessed directly by implanting microelectrode arrays in the dorsal root ganglia (DRG), which provide a compact and stable target for stimulating a diverse group of sensory fibers. To gain insight into factors affecting the number and types of primary afferents activated, we developed a computational model that simulates the recruitment of fibers in the feline L7 DRG. The model comprises two parts. The first part is a single-fiber model used to describe the current-distance relation and was based on the McIntyre-Richardson-Grill model for excitability. The second part uses the results of the singe-fiber model and published data on fiber size distributions to predict the probability of recruiting a given number of fibers as a function of stimulus intensity. The range of intensities over which exactly one fiber was recruited was approximately 0.5-5 µA (0.1-1 nC per phase); the stimulus intensity at which the probability of recruiting exactly one fiber was maximized was 2.3 µA. However, at 2.3 µA, it was also possible to recruit up to three fibers, albeit with a lower probability. Stimulation amplitudes up to 6 µA were tested with the population model, which showed that as the amplitude increased, the number of fibers recruited increased exponentially. The distribution of threshold amplitudes predicted by the model was similar to that previously reported by in vivo experimentation. Finally, the model suggested that medium diameter fibers (7.3-11.5 µm) may be recruited with much greater probability than large diameter fibers (12.8-16 µm). This model may be used to efficiently test a range of stimulation parameters and nerve morphologies to complement results from electrophysiology experiments and to aid in the design of microelectrode arrays for neural interfaces.

Sensitivity of 5-fluorouracil-resistant cancer cells to adenovirus suicide gene therapy.

A promising approach for cancer gene therapy is the combination of adenovirus vectors (AdV) with the suicide gene cytosine deaminase and uracil phosphoribosyl transferase (CDColon, two colonsUPRT). While such vectors have been tested in tumor cell lines and xenograft models, it is not clear how these therapeutic vectors would perform in primary human tumors. We, thus, examined the effect of the combination of a recombinant adenovirus expressing the CDColon, two colonsUPRT (AdCU) with 5-fluorocytosine (5-FC) on primary cancer cells isolated from the ascites or pleural fluids of patients with metastatic cancers. In such models, we have found a direct correlation between the patients' response to 5-FU and the response shown by the cancer cells in vitro, confirming the clinical relevance of this methodology. Our findings demonstrated that this combination was able to kill primary tumor cells, including those that had developed resistance to 5-FU. Furthermore, while proliferating cells were more susceptible to 5-FU, the combination was effective in both rapid and slow proliferating samples. Our study demonstrated that this gene therapy approach could provide an effective therapeutic option for cancers and is not affected by acquired 5-FU resistance. Also of importance is the effectiveness of this gene therapy approach on slower proliferating cells that is typical of the majority of cancers in vivo. This suggests a greater likelihood that it will be effective in a clinical setting.

Characterization of elongation factor-1A (eEF1A-1) and eEF1A-2/S1 protein expression in normal and wasted mice.

The eEF1Alpha-2 gene (S1) encodes a tissue-specific isoform of peptide elongation factor-1A (eEF1A-1); its mRNA is expressed only in brain, heart, and skeletal muscle, tissues dominated by terminally differentiated, long-lived cells. Homozygous mutant mice exhibit muscle wasting and neurodegeneration, resulting in death around postnatal day 28. eEF1Alpha-2/S1 protein shares 92% identity with eEF1A-1; because specific antibodies for each were not available previously, it was difficult to study the developmental expression patterns of these two peptide elongation factors 1A in wasted and wild-type mice. We generated a peptide-derived antiserum that recognizes the eEF1Alpha-2/S1 isoform and does not cross-react with eEF1A-1. We characterized the expression profiles of eEF1A-1 and eEF1A-2/S1 during development in wild-type (+/+), heterozygous (+/wst), and homozygous (wst/wst) mice. In wild-type and heterozygous animals, eEF1A-2/S1 protein is present only in brain, heart, and muscle; the onset of its expression coincides with a concomitant decrease in the eEF1A-1 protein level. In wasted mutant tissues, even though eEF1A-2/S1 protein is absent, the scheduled decline of eEF1A-1 occurs nonetheless during postnatal development, as it does in wild-type counterparts. In the brain of adult wild-type mice, the eEF1A-2/S1 isoform is localized in neurons, whereas eEF1A-1 is found in non-neuronal cells. In neurons prior to postnatal day 7, eEF1A-1 is the major isoform, but it is later replaced by eEF1A-2/S1, which by postnatal day 14 is the only isoform present. The postdevelopmental appearance of eEF1A-2/S1 protein and the decline in eEF1A-1 expression in brain, heart, and muscle suggest that eEF1A-2/S1 is the adult form of peptide elongation factor, whereas its sister is the embryonic isoform, in these tissues. The absence of eEF1A-2/S1, as well as the on-schedule development-dependent disappearance of its sister gene, eEF1A, in wst/wst mice may result in loss of protein synthesis ability, which may account for the numerous defects and ultimate fatality seen in these mice.

Rapid up-regulation of peptide elongation factor EF-1alpha protein levels is an immediate early event during oxidative stress-induced apoptosis.

Hydrogen peroxide (H(2)O(2)) induces apoptosis in cultured cells, in a dose-dependent manner. Treatment with H(2)O(2) causes decreased mitochondrial respiration, along with DNA degradation and the formation of an oligonucleosomal ladder, all hallmarks of apoptotic cell death. In this report, we investigate alterations in expression of a peptide elongation factor, EF-1alpha, during oxidative challenge. EF-1alpha protein levels undergo rapid increase upon treatment with H(2)O(2); however, whereas sublethal doses of H(2)O(2) stimulate only transient increases of EF-1alpha protein levels, lethal doses produce sustained elevation of EF-1alpha levels. Furthermore, pretreatment of H9c2(2-1) cells with transcriptional inhibitors fails to abolish the oxidant-induced increase in EF-1alpha, and Northern blotting analysis reveals that EF-1alpha mRNA levels remain steady throughout the H(2)O(2) treatment period, suggesting that the up-regulation of EF-1alpha is mediated posttranscriptionally. Transient transfection with an antisense EF-1alpha cDNA protects against hydrogen peroxide-mediated cytotoxicity in proportion to the degree of repression of EF-1alpha protein levels, suggesting that up-regulation of EF-1alpha plays a role in expediting the execution of the apoptotic program in response to oxidative stress.

Apoptosis rate can be accelerated or decelerated by overexpression or reduction of the level of elongation factor-1 alpha.

Peptide chain elongation factor-1 alpha (EF-1 alpha) is required for the binding of aminoacyl-tRNAs to acceptor sites of ribosomes during protein synthesis. More recently, EF-1 alpha has been shown to be involved in cytoskeletal organization. The elongation factor functions in actin bundling and microtubule severing. Moreover, it can activate the phosphatidylinositol-4 kinase whose substrates are involved in regulation of actin polymerization. The expression level of EF-1 alpha is regulated in many situations such as growth arrest, transformation, and aging. Because of this regulation of EF-1 alpha in various states of cell life, and its key position in protein synthesis as well as cytoskeletal organization, we chose to investigate the effect of its expression levels on apoptosis. Apoptosis is a complex event regulated through numerous activators and inhibitors. In some situations, protein synthesis is required for apoptosis to be triggered. Investigation of the effect of altered levels of elongation factor-1 alpha on apoptosis is of particular interest since it may affect both protein synthesis and cytoskeletal organization. For example, reduction of EF-1 alpha leads to a reduced protein synthesis rate, which might reduce the presence of those "killer factors" triggering apoptosis. EF-1 alpha involvement in cytoskeletal organization is another example, since cytoskeletal organization undergoes dramatic changes during apoptosis. Thus, this study has been planned to ascertain whether hypo- and hyperexpression of EF-1 alpha protein, achieved by constructing expression vectors with the EF-1 alpha cDNA in its antisense or sense orientation under the control of a cytomegalovirus promoter, can produce stable transfectants with either heightened or reduced responsiveness to apoptosis stimuli. Our results show the following: (1) induction of apoptosis by serum deprivation shows that antisense EF-1 alpha provides cells significant protection from apoptotic cell death and (2) EF-1 alpha overexpression causes a faster rate of cell death. These findings suggest that when EF-1 alpha protein is abundant the cells are proapoptosis, and vice versa in low abundance the cells are in the mode of antiapoptosis. Therefore, changes in levels of EF-1 alpha may be one of the global pivotal regulators modulating the rate of apoptosis.

Oligodendrocyte maturation and progenitor cell proliferation are independently regulated by thyroid hormone.

The development of oligodendrocyte progenitor cells is regulated by epigenetic factors which control their proliferation and differentiation. When oligodendrocyte progenitor cells, purified on a Percoll centrifugation gradient from neonate rat brain, are cultured in serum-free medium in the presence of platelet-derived-growth factor (PDGF), they divide and their differentiation is delayed. Triiodothyronine (T3) treatment of progenitor cells blocks their proliferation and induces their differentiation into oligodendrocytes. T3 also induces morphological differentiation of oligodendrocytes as indicated by the marked increase in the length of oligodendrocyte processes. To determine whether the effects of T3 on progenitor cell proliferation and oligodendrocyte maturation are causally related, or instead, are independent, we examined the influence of T3 on secondary cultures of postmitotic oligodendrocytes. We show that T3 increases morphological and functional maturation of postmitotic oligodendrocytes as indicated by a well developed network of branched processes and by the expression of myelin/oligodendrocyte glycoprotein (MOG) and glutamine synthetase (GS). T3 increases glutamine synthetase activity and its message level after a lag period of 24-48 h, and these levels increase through a posttranscriptional event. In contrast, no effect of T3 was observed on myelin basic protein (MBP) gene expression as determined by Northern blot analysis. Our results indicate that thyroid hormones participate in the control of the progenitor cell proliferation and differentiation as well as in oligodendrocyte maturation and that these two T3-regulated events are independent.

Secondary structures and features of the 18S, 5.8S and 26S ribosomal RNAs from the Apicomplexan parasite Toxoplasma gondii.

The two major subunits of the ribosomal RNA (rRNA) of Toxoplasma gondii, 18S and 26S, as well as 5.8S, have been sequenced and folded according to known consensus and established secondary structures. Conserved and variable nucleotide (nt) regions were identified using multiple alignments with rRNA sequences of selected organisms. The 18S rRNA showed a well conserved core structure of 48 stems and a hypervariable V4 region identified four additional stems including a pseudoknot. The 18S rRNA contained an additional helix in the V2 region located between nt 204 to 258. We noted that T. gondii 18S does not have a true V6 region, but was organized as a motif of a simple stem. T. gondii 26S had a conserved core structure of 83 stems and its expansion segments, so-called divergent domains, demonstrated a high degree of similarity with secondary structures from rRNA of dinoflagellates and ciliates. For the T. gondii 26S sequence, we found two additional stems, D3d and D3e, composed of 140 nt having a higher deltaG value. These segments are absent from the prokaryotic rRNA structures, whereas the hypervariable V4 region of the small subunit is not as variable. The well preserved structures could indicate an additional function for the eukaryotic ribosome.

Toxoplasma gondii: structure and characterization of the 26S ribosomal RNA and peptidyl transferase domain.

The 26S, 5.8S, and the intergenic spacer ribosomal DNAs (rDNA) of Toxoplasma gondii have been cloned and completely sequenced from both DNA strands. The length of the large subunit was found to be 3487 nucleotides and the 5.8S was 153 nucleotides long. These formed the large rRNA subunit of Toxoplasma and were mapped in the rDNA unit known to be repeated 110 times in a head-to-tail fashion in the genome. Primer extension analysis and multiple alignments localized the 5' end point of the two rRNAs. Comparisons with Toxoplasma rDNA by nucleic acid homology studies gave 76% similarity with the dinoflagellate Prorocentrum micans, 66% with yeast Saccharomyces cerevisiae, and 64% with the ciliate Tetrahymena thermophila. Similarity was apparent in the conserved core structure of the large subunit rRNA and divergent sequences were identified in the so-called divergent domains. Construction of a secondary structure model of the peptidyl transferase center of the large rRNA revealed similarities with the same domain from other life forms.

Expression of alpha and beta thyroid receptors during oligodendrocyte differentiation.

To determine which thyroid receptor (TR) isoforms are expressed during oligodendrocyte differentiation, we studied the expression of the mRNAs encoding two alpha (alpha 1 and alpha 2) and one beta (beta 1) TR isoforms in a bipotential oligodendrocyte-type 2-astrocyte (O-2A) progenitor cell line (CG-4) as well as in rat O-2A progenitor cells and oligodendrocytes. O-2A progenitor cells expressed only TR alpha-mRNAs, whereas oligodendrocytes and type 2-astrocytes also expressed TR beta 1-mRNAs. The differential expression of alpha 1 and beta 1 TRs suggests specific functions for both types of TRs during oligodendrocyte differentiation. We present evidence for a possible role of TR alpha 1 in the effect of thyroid hormones on the proliferation of CG-4 cells maintained as progenitor cells.

[Geriatric digestive surgery. An analysis of 163 cases]. / Chirurgie digestive gériatrique. Une analyse de 163 cas.

The aim of our study is to analyze the clinical features and outcome of digestive surgery in the aged. From Jan. 1979 to Dec. 1981, 1,389 operations under general anesthesia were performed on patients 75 years old and more. From this group, 163 patients (111 females and 52 males, mean age: 79 +/- 0.7 years) underwent surgery of the alimentary tract. The procedures were divided in: colorectal (48%), biliary (32%), gastric (10%), small bowel (6%), esophagus (1%) and others (3%). An operation for cancer was performed in 63 patients: palliative (69%), curative (31%). The mean length of hospital stay is 23 days (pre-op 7 days, post-op 16 days). Only 16% of the patients needed intensive care. Postoperative complications occurred in 43 patients (26%); cardiovascular (47%), psychiatric (26%), pulmonary (23%) and others (4%). The overall mortality rate is 10%: 6% for elective cases and 24% for urgent cases (49 patients). The mortality is related to: sepsis and peritonitis (53%), cardiopulmonary disease (23%), hemorrhage (12%), cachexia (12%). At discharge, 62% of the patients returned home directly, 18% to convalescent homes, 10% to unknown places and 10% in nursing homes. Our data supports the benefit of surgery in the aged.

Pancreatic carcinoma: surgical treatment.

There is controversy in the literature over the surgical treatment of pancreatic carcinoma, especially whether gastro-enterostomy should be performed prophylactically when palliative biliary bypass is done. The authors report a retrospective study of 184 patients with pancreatic carcinoma treated at the Hôtel-Dieu Hospital in Montreal between 1970 and 1982. Of these, 106 patients had cancer of the head of the pancreas. In this group, 15 had a Whipple procedure. Median survival was 512 days with no 5-year survivors. Forty-nine patients had biliary bypass alone and 3 needed gastroenterostomy later for duodenal obstruction. Eleven patients had both biliary--enteric bypass and gastroenterostomy. Biliary bypass using the common duct gives the same result as that using the gallbladder. Considering their reoperation rate of only 7.3%, the authors do not believe that prophylactic gastroenterostomy is indicated in these patients.

Demonstration of a bronchobiliary fistula by 99mTc-HIDA cholescintigraphy.

A case of bronchobiliary fistula diagnosed by 99mTc-HIDA cholescintigraphy is presented. The fistula caused by a stenosing tumor of the left hepatic duct would probably have been missed without the use of delayed views and body fluids counting which increased the specificity of scintigraphic findings.

Arteriography and pancreatic disease.

In the last 10 years, 605 celiomesenteric arteriographic examinations were done at Hôtel-Dieu de Montréal, including 236 for suspected pancreatic disease. Sixty patients with histologic proof of malignant neoplasm had a positive arteriographic examination while 136 had benign disease of the pancreas and 40 others had extra-pancreatic disease. In this series, the sensitivity of arteriography was 88.3% and the specificity 92.6%. In the last 5 years the sensitivity of superselective arteriography was 100%. These results were better than those obtained with noninvasive techniques obtained with noninvasive techniques such as computerized axial tomography, ultrasonography and duodenography. The authors conclude that arteriography and particularly superselective arteriography is an excellent method for evaluating patients with suspected pancreatic cancer.

Computerized axial tomography and fine-needle biopsy in surgery of the pancreas.

Notable advances have been made in the investigation of pancreatic disorders. However, many of the diagnostic tests required are still of an invasive nature and are attended by difficulty and risk. The authors have found that the noninvasive method of axial tomography is highly reliable for the evaluation of pancreatic lesions, especially in cases in which a pancreatic mass is present or the duct of Wirsung is dilated. This procedure can also be helpful in the follow-up of patients who have suffered from acute or chronic pancreatitis. The same diagnostic problem is present at operation. To obtain a quick and reliable diagnosis of pancreatic lesions, the authors performed fineneedle aspiration biopsies followed by immediate staining and examination of the specimen. Among 43 patients an accurate diagnosis was obtained in 41 instances and no complication was ascribed to this diagnostic procedure.