Association between brain temperature and dentate field potentials in exploring and swimming rats.

Attempts to correlate behavioral learning with cellular changes, such as increased synaptic efficacy, have often relied on increased extracellular potentials as an index of enhanced synaptic strength. A recent example is the enlarged excitatory field potentials in the dentate gyrus of rats that are learning spatial relations by exploration. The altered hippocampal field potentials do not reflect learning-specific cellular changes but result from a concomitant rise in brain temperature that is caused by the associated muscular effort. Enhanced dentate field excitatory potentials followed both passive and active heating and were linearly related to the brain temperature. These temperature-related effects may mask any learning-induced changes in field potential.

Synchronized Ca2+ oscillations induced in Madin Darby canine kidney cells by bradykinin and thrombin but not by ATP.

In an earlier report, we described synchronous Ca2+ oscillations in globally stimulated, subconfluent MDCK cells [Røttingen J-A, Enden T., Camerer E., Iversen J-G., Prydz H. Binding of human factor VIIa to tissue factor induces cytosolic Ca2+ signals in J82 cells, transfected COS-1 cells, Madin-Darby canine kidney cells and in human endothelial cells induced to synthesize tissue factor. J Biol Chem 1995; 270: 4650-4660]. In order to elucidate the mechanisms behind these oscillations, we have analyzed the fluctuations in cytosolic Ca2+ in single, Fura-2 loaded, MDCK cells grown to subconfluence, after stimulation with bradykinin, thrombin and ATP. All three agonists gave rise to an initial Ca2+ spike followed by oscillations or transients. Both the initial and subsequent spikes appeared to be due mainly to release of Ca2+ from internal stores, since they remained after Ca2+ influx was impeded by either La3+ or by chelation of extracellular Ca2+ with EGTA. The secondary spikes were apparently synchronized when the cells were (permanently and globally) stimulated with bradykinin or thrombin, but each cell seemed to oscillate independently when stimulated in the same way with ATP. Synchronized secondary spikes arose with a constant frequency and amplitude, independent of agonist concentration in contrast to most Ca2+ oscillations observed. Pretreatment of the cells with octanol to block gap junctions, or with EGTA or La3+ to inhibit Ca2+ influx, abolished the synchronization induced by bradykinin or thrombin. We observed that in the MDCK cell layer there are some "pacemaker' cells and hypothesize that these have a higher sensitivity for the agonists than their neighboring cells. From these pacemakers, an intercellular Ca2+ wave can be seen to spread to adjacent cells in the presence of intact gap junctions, thereby initiating concurrent transients in all cells. The Ca2+ wave is amplified by release from internal stores, probably owing to the bell-shaped Ca2+ activation curve of the IP3 receptor and by subsequent Ca2+ influx through Ca2+ release activated channels.

Differential expression and regulation of the PKA signalling pathway in fast and slow skeletal muscle.

To identify intracellular signalling pathways that transduce muscle electrical activity, we have investigated the Protein Kinase A (PKA) pathway in fast and slow skeletal muscle. The slow soleus muscle (SOL) displayed approximately twice as much PKA catalytic activity and cAMP-binding compared to the fast Extensor Digitorum Longus (EDL) muscle. These results were confirmed by Western blot analysis using antibodies directed against the catalytic or regulatory subunits of PKA. PKA subunits were concentrated at the neuromuscular junction in innervated and denervated muscle fibers demonstrating that PKA is expressed post-synaptically. In addition, we also detected PKA subunits outside the junctional area, suggesting that PKA functions outside of the synaptic regions. Following denervation, levels of cyclic AMP, PKA C activity, R cAMP-binding and RI alpha protein levels increased significantly in the SOL, in contrast to the EDL where only elevated levels of RI alpha protein were observed. These observations demonstrate that PKA levels in skeletal muscle are subject to control at several levels and suggest that some of the differences may be in the pattern of electrical activity that motoneurons impose on the SOL and EDL.

[Nonpenetrating deep trabeculectomy treated with mitomycin C, without implant. A prospective evaluation of 55 cases]. / Trabéculo-sclérectomie non perforante avec mitomycine C, sans implant. Evaluation prospective sur 55 cas.

PURPOSE: Since 1998 we have been conducting a prospective study of nonpenetrating deep trabeculectomy with chronic open-angle glaucoma to evaluate the efficiency of the technique. MATERIAL AND METHODS: The study was carried out in 55 eyes of 41 patients who suffered from open-angle glaucoma. After performing a superior scleral flap, mitomycin diluted to 0.01% was applied for 3 minutes, then the 4 x 4-mm superficial scleral flap was dissected at two-thirds deepness until reaching the cornea. The Schlemm canal and the external trabecula were surgically removed and the two points of the Schlemm canal were catheterized with a trabeculotome to ensure that the ablation was well done. If it was not, it was completed by using a trabeculotome as a guide. Postoperatively, if the filtering bleb tended to decrease or ocular pressure began to increase, the operated trabecular region was reopened with Yag laser. The filtering bleb characteristics were correlated with the normalization of intraocular pressure in the first 30 cases. RESULTS: Preoperative pressure without treatment was 32 mmHg. Postoperative intraocular pressure without treatment was 20 mmHg or less in 79% of the eyes after 4 months, 77.5% after 6 months, 75% after 8 months and 61% after 12 months. By adding a local hypotension treatment in monotherapy, a pressure of 20 mmHg or less was obtained in 79% of the cases after 12 months. No severe complications were observed. The presence of a filtering bleb is an important factor in the normalization of postoperative pressure (p=0.0048). CONCLUSIONS: This surgical technique provides a substantial decrease in intraocular pressure and very few complications after 12 months of follow-up.

Electropermeabilization of skeletal muscle enhances gene transfer in vivo.

This work demonstrates that electrical muscle stimulation markedly increases the transfection efficiency of an intramuscular injection of plasmid DNA. In soleus or extensor digitorum longus muscles of adult rats the percentage of transfected fibers increased from about 1 to more than 10. The number of transfected fibers and the amount of foreign protein produced could be graded by varying the number or duration of the electrical pulses applied to the muscle. The stimulation had to be applied when DNA was present in the muscle. When dextran was injected together with the plasmid DNA, it was also taken up by the transfected fibers. Stimulation-induced membrane permeabilization and increased DNA uptake were therefore probably responsible for the improved transfection. The stimulation caused some muscle damage but the fibers regenerated rapidly. The described method, which is simple, efficient, and reproducible, should become valuable for basic research, gene therapy and DNA vaccination.

gamma-AChR/epsilon-AChR switch at agrin-induced postsynaptic-like apparatus in skeletal muscle.

We transfected the extrajunctional region of denervated soleus muscles in adult rats with neural agrin cDNA to induce myofibers to form postsynaptic-like apparatus containing acetylcholine receptor (AChR) aggregates. By 1 week approximately 30% of the AChR aggregates contained a mixture of epsilon-AChRs and gamma-AChRs while approximately 70% had only gamma-AChRs. If the transfected muscles were reinnervated in the original junctional region, the postsynaptic-like apparatus, despite the absence of apposed axon terminals, gradually came to have only epsilon-AChRs. We conclude that at the postsynaptic apparatus of ectopic neuromuscular junctions formed by a foreign nerve implanted into the extra-junctional region of denervated muscles, agrin secreted by the axon terminal plays a direct role in the gamma-AChR/epsilon-AChR switch that occurs as the apparatus reaches maturity. Our findings, together with results from other studies, indicate further that agrin and acetylcholine are the only nerve-derived factors required for this switch.

Regulation of the size and distribution of agrin-induced postsynaptic-like apparatus in adult skeletal muscle by electrical muscle activity.

We compared actylcholine receptor (AChR) aggregates induced by neural agrin released from transfected muscle fibers with AChR aggregates induced by transplanted axons in extrajunctional regions of denervated rat soleus muscles. Both neural agrin and transplanted axons induced multiple, irregularly distributed AChR aggregates on muscle fibers. Direct electrical muscle stimulation of transfected muscles for up to 10 weeks removed all agrin-induced AChR aggregates (the losers) except one (the winner) on many fibers. Axon-induced AChR aggregates underwent comparable selection of winners and losers. The results suggest that agrin and acetylcholine-driven muscle activity provided by transplanted axons are sufficient to elicit in a denervated adult muscle fiber processes that regulate the size and distribution of ectopic neuromuscular junctions.

In vivo electroporation improves immune responses to DNA vaccination in sheep.

In vivo electroporation was utilised to enhance plasmid DNA expression in sheep muscle to improve the immune response to DNA vaccination. DNA encoding enhanced green fluorescence protein expressed at higher levels in sheep muscle following in vivo electroporation which caused minimal muscle damage. Groups of seven sheep were then given three intramuscular injections of plasmids encoding two Haemonchus contortus Ag, with and without electroporation at 0, 3 and 7 weeks. Humoral responses were enhanced in electroporated sheep. Four weeks after vaccination, all groups were injected subcutaneously with recombinant Ag formulated in Quil A. Induction of vaccine-specific immune memory was demonstrated in DNA-vaccinated sheep.

Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation.

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 microgram of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.

Long-term cerebral effects of CNS chemotherapy in children with acute lymphoblastic leukemia.

Fifteen 7-16-year-old patients, treated in 1981-1984 for acute lymphoblastic leukemia (ALL) in first complete remission, were studied. As a central nervous system prophylaxis, all the children were treated with repeated methotrexate (MTX) instillations, but none were irradiated. The study protocol included magnetic resonance (MR) and a battery of neuropsychological tests. Small, punctate white-matter lesions were found by MR in eight children, probably minor vascular lesions. All the children were within normal intelligence range with a mean total WISC-R IQ of 109. Minor neuropsychologic problems were found in two patients, while one child showed a more extensive specific learning disorder in school.

DNA injection in combination with electroporation: a novel method for vaccination of farmed ruminants.

Injection of plasmid DNA encoding antigens into rodents followed by electroporation improved the immune response when compared with injection without electroporation (Widera et al. J Immunol 2000;164:4635-40; Zucchelli et al. J Virol 2000;74:11598-607; Kadowaki et al. Vaccine 2000;18:2779-88). The present study describes the extension of this technology to farm animals, by injecting plasmid DNA encoding mycobacterial antigens (MPB70, Ag85B and Hsp65) into the muscles of goats and cattle using two different types of electrodes, both allowing DNA delivery at the site of electroporation. The animals were vaccinated under local anaesthesia without any observed immediate or long-term distress or discomfort, or any behavioural signs of muscle damage or pathological changes after the electroporation. DNA-injected and electroporated goats showed increased humoral response after the primary vaccination when compared with nonelectroporated animals. Improved T-cell responses following electroporation were observed in hsp65 DNA-vaccinated cattle. DNA injection with or without electroporation did not compromise the specificity of the tuberculin skin test. In conclusion, a protocol applying in vivo electroporation free of side effects to farmed ruminants was established. In addition, we show that DNA vaccination in combination with electroporation can improve the primary immune responses to the encoded antigens.

Childhood non-Hodgkin's lymphoma in the five Nordic countries. A five-year population-based study from the Nordic Society of Pediatric Hematology and Oncology.

PURPOSE: The comparable health-care organizations and common Cancer Registry for childhood malignancies in the five Nordic countries offered an opportunity to conduct an epidemiological study on a reasonable number of childhood non-Hodgkin's lymphoma (NHL) cases collected in a population-based manner. MATERIAL AND METHODS: All childhood cases (0-14.9 years at diagnosis) reported during the 5-year period of 1985-1989 to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) Cancer Registry for childhood malignancies were reviewed and analyzed according to age, Murphy's stage, gender, site, and survival. RESULTS: The annual incidence of NHL is 0.7 per 100,000 children in the five Nordic countries, constituting 5% of all childhood malignancies. Age distribution was even; the male/female ratio was 3:1. Age and stage were shown by Cox regression analysis to be independent prognostic factors. Older age and lower stage affected outcome favorably. The stage and site distribution was similar to previous reports. Survival data were in accordance with those expected with modern treatment protocols. CONCLUSIONS: The incidence and relative frequency of NHL in childhood in the five Nordic countries is in agreement with previously reported data, but the even distribution of cases throughout childhood is a new finding. Older age at onset and stage of disease affect outcome favorably, whereas male gender contrary to acute lymphoblastic leukemia was not found to affect outcome.

Spectrum of perforin gene mutations in familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive disease of early childhood characterized by nonmalignant accumulation and multivisceral infiltration of activated T lymphocytes and histiocytes (macrophages). Cytotoxic T and natural killer (NK) cell activity is markedly reduced or absent in these patients, and mutations in a lytic granule constituent, perforin, were recently identified in a number of FHL individuals. Here, we report a comprehensive survey of 34 additional patients with FHL for mutations in the coding region of the perforin gene and the relative frequency of perforin mutations in FHL. Perforin mutations were identified in 7 of the 34 families investigated. Six children were homozygous for the mutations, and one patient was a compound heterozygote. Four novel mutations were detected: one nonsense, two missense, and one deletion of one amino acid. In four families, a previously reported mutation at codon 374, causing a premature stop codon, was identified, and, therefore, this is the most common perforin mutation identified so far in FHL patients. We found perforin mutations in 20% of all FHL patients investigated (7/34), with a somewhat higher prevalence, approximately 30% (6/20), in children whose parents originated from Turkey. No other correlation between the type of mutation and the phenotype of the patients was evident from the present study. Our combined results from mutational analysis of 34 families and linkage analysis of a subset of consanguineous families indicate that perforin mutations account for 20%-40% of the FHL cases and the FHL 1 locus on chromosome 9 for approximately 10%, whereas the major part of the FHL cases are caused by mutations in not-yet-identified genes.