ATP-gated current in dissociated rat nucleus solitarii neurons.

1. The excitatory response of extracellularly applied ATP was investigated in freshly dissociated rat nucleus tractus solitarii neurons under whole-cell configuration using the "concentration-clamp" technique. 2. At a holding potential of -70 mV, 100 microM ATP evoked inward current that was slowly desensitized in the continuous presence of ATP. The ATP-gated current increased in a concentration-dependent manner over the concentration range between 10 microM and 1 mM. The half-maximum concentration was 31 microM and the Hill coefficient was 1.2. 3. The potency of ATP analogues for the purinergic receptor was in the order of ATP = 2-methylthio-ATP much greater than ADP greater than alpha,beta-methylene ATP. Neither adenosine nor AMP evoked any responses. The order was consistent with a P2y receptor subtype. 4. The current-voltage relationship for the 100 microM ATP response showed a clear inward rectification at positive potentials beyond -50 mV. The reversal potential of the ATP-gated current was +13 mV. 5. The time constants of activation and inactivation of the ATP-gated current solution were dependent on the extracellular ATP concentration, and both kinetics became faster at higher ATP concentrations. 6. The ATP-gated current was also elicited in an external solution containing Ca2+ as a permeable cation. The inactivation kinetics in an external solution containing 75 mM Ca2+ were faster than those in an external solution with 150 mM Na+. 7. Calculated relative permeability ratios were PNa/PCs = 1.64 ([Na+]o = 30-150 mM), PCa/PCs = 2.17 ([Ca2+]o = 2 mM). Anions were not measurably permeable in this preparation.     

Increased frequency of IFN-gamma-producing peripheral CD8+ T cells with memory-phenotype in patients with chronic hepatitis C

To identify the capacity for cytokine production and the phenotypic characteristics of peripheral CD8(+) T cells in patients with chronic hepatitis C, 31 patients with chronic hepatitis C and 22 healthy controls were studied at the single cell level by three-color flow cytometry. Whole blood was stained with surface CD8, intracellular interferon-gamma (IFN-gamma), and interleukin-4 (IL-4), surface CD8, CD28, and intracellular IFN-gamma after stimulation with PMA plus ionomycin, and then surface CD8, CD45RA, and CD28. IFN-gamma-producing peripheral CD8(+) T cells were found frequently in patients than in controls (P < 0.05), whereas IL-4-producing peripheral CD8(+) T cells were not. Although the frequency of peripheral CD28(+)CD8(+) and CD28(-)CD8(+) T cells in patients was not different from that of controls, CD28(+)CD8(+) T cells exceeded CD28(-)CD8(+) T cells in the capacity for IFN-gamma-production after mitogenic stimulation (P < 0.01). In a more detailed analysis of the CD28(+)CD8(+) T cells, CD45RA(-)CD28(+)CD8(+) T cells, defined phenotypically as memory cells, were found frequently in patients than in controls (P < 0.05). There were no significant correlations between the frequency of IFN-gamma-producing peripheral CD8(+) T cells and hepatitis C virus RNA level or serum alanine aminotransferase level in patients. These data suggest that functionally T cytotoxic type 1 and memory CD8(+) T cells are predominant in the peripheral blood of chronic hepatitis C patients and that such activated CD8(+) T cells are associated with liver damage.     

Incidence of anti-DNA idiotype-positive cells in human peripheral blood

Human anti-DNA idiotype (Id)-bearing cells in peripheral blood were sought using mouse monoclonal anti-Id antibodies to human monoclonal anti-DNA antibodies. Pretreatment with acid pH or pronase P or preincubation in human serum-free medium markedly decreased the number of anti-Id-reactive cells. Pronase P-treated cells were able to bind to anti-Id once more after incubation for 18 hr at 37° C, indicating the resynthesis of internal idiotypic determinants on the cells. Antiidiotype-reactive cells retained the same idiotypes in their cytoplasma. The cells expressing anti-DNA idiotypes, termed 0-81 and NE-1, were detected in the circulation of most patients with active lupus nephritis but not in those of inactive systemic lupus erythematosus (SLE) and healthy subjects. The idiotype-positive cells occurred in up to 5–10% of B cells from some patients with SLE in the active stage but became undetectable in remission. A limited number of anti-DNA Id-positive cells responsible for anti-DNA production might be preferentially expanded during acute episodes of the disease in some patients with SLE.     

Liver Damage in Hemodialysis Patients with Hepatitis C Virus Viremia: A Prospective 10-Year Study

Hepatitis C virus (HCV) infection is a major problem associated with hemodialysis. The extent of liver damage in hemodialysis patients with chronic HCV infection has not been thoroughly documented. The aim of this study was to evaluate liver damage of hemodialysis patients infected with HCV. A total of 233 hemodialysis patients were categorized into two groups at entry: group X, 80 positive for serum HCV RNA, and group Y, 153 negative for serum HCV RNA. All were tested for serum alanine aminotransferase (ALT) serially from 1989 to 1998, and serum hyaluronic acid (HA), serum type-IV collagen (IV-C), platelet counts, and ultrasonographic examination of the liver was done in 1998. In group X, 61.3% had continuously abnormal ALT levels for over six months followed by normal ALT levels. Of the group X patients, 11.3% had abnormal ALT levels in 1998, and in three, hepatocellular carcinoma occurred. Mean HA and IV-C levels in group X (648.8 and 188.7 ng/ml, respectively) were significantly higher than in group Y (213.1 and 165.5 ng/ml, respectively) (P < 0.05). Ultrasonographic findings significantly correlated with serum HA level and platelet counts and showed significantly more abnormalities in group X than in group Y (P < 0.05). From these findings, a combined examination with ultrasonography and serum fibrogenesis markers is useful for detection of liver damage in hemodialysis patients with HCV viremia.     

Osteoarthritis of the atlanto-axial joint

Summary Thirty-one patients suffering from osteoarthritis of the atlanto-axial joint are described, suggesting that this disease is not particularly uncommon.The most characteristic radiological signs are narrowing of the joint space from loss of articular cartilage, marginal cortical thickening and osteophyte formation. Histological examination in the cadaver has shown thinning and fibrillation of articular cartilage and intra-cartilaginous ossification.The radiological appearances of the atlanto-axial joint are classified in three types, lateral atlanto-axial, atlanto-odontoid and mixed. Peak age distribution occurs between 50 and 69 years of age. Patients complain of occipital pain, a stiff shoulder and paraesthesia of the fingers. Treatment was conservative in all cases except one, a 63-year-old woman with severe occipital pain. A transoral atlanto-axial fusion was performed in this case with marked improvement. The transoral approach allows fixation and curettage of the degenerative lesions.

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Résumé L'ostéoarthrite de l'articulation atloïdo-axoïdienne n'a été que rarement décrite. L'auteur en a néanmoins réuni 31 observations ce qui prouve que cette affection n'est pas exceptionnelle. Les signes radiologiques les plus caractéristiques sont le pincement de l'interligne articulaire dû à l'amincissement du cartilage, l'épaississement cortical marginal et l'ostéophytose. L'examen microscopique des pièces cadavériques a montré un amincissement et un aspect fibrillaire du cartilage articulaire ainsi que l'existence d'ossifications intracartilagineuses.L'atteinte radiologique de l'articulation atloïdo-axoïdienne a été classée en trois types: atloïdo-axoïdien latéral, atloïdo-odontoïdien et mixte, associant les deux lésions.Le maximum de fréquence se situe entre 50 et 69 ans. Les malades qui se plaignaient d'occipitalgies, de raideur des épaules et de paresthésie des doigts ont été traités par les méthodes habituelles, mais dans le cas d'une femme de 63 ans qui souffrait d'occipitalgie sévère, on a réalisé la fusion trans-orale de l'articulation atloïdo-axoïdienne et obtenu une notable amélioration.La voie d'accès trans-orale de l'articulation atloïdo-axoïdienne peut donc être utilisée dans de tels cas pour le curetage des lésions scléreuses et kystiques et la fixation.

     

One-bone forearm formation using vascularized fibula graft for massive bone defect of the forearm with infection: case report

Massive long-bone defects of greater than 6 cm are difficult to treat with conventional bone grafts, and other methods are sometimes recommended, such as vascularized bone grafts or bone transport using the Ilizarov external fixator. The combination of local infection with a massive bone defect exacerbates the problem, and provides an even more negative prognosis. The authors treated a large bone defect of the forearm with local infection, using a one-bone forearm formation with a large vascularized fibula graft. They attached an adequate amount of muscle fascia to the vascularized fibula, which was useful not only for coverage of the skin defect, but also for treatment of the local infection. Twenty months after surgery, elbow and hand functions were maintained, and the patient had no disturbance of hand function in daily activities, although rotation of the forearm was sacrificed.     

Combining free vascularized fibula graft and the Ilizarov external fixator: recent approaches to congenital pseudarthrosis of the tibia

The present study compares a recent approach for congenital pseudarthrosis of the tibia, combining a free vascularized fibula graft and an Ilizarov external fixator. Between 1984 and 1993, seven vascularized fibula grafts were performed. In the first six cases, casts or unilateral external fixators were applied, and five of these cases were complicated by fractures of the graft, necessitating secondary procedures or long-term immobilization. The mean follow-up was 12.1 years. Five patients can now walk unassisted, and one uses a brace only outdoors. On average, a brace was required for 18 months. In the recent Ilizarov external fixator case, the fixator was removed after 4 months and a brace was unnecessary at 10 months. There was no leg-length discrepancy, severe ankle deformity, or fracture of the grafted fibula 7 years postoperatively. Combining a free vascularized fibula graft with an Ilizarov external fixator is recommended for this condition.     

Limited numbers of recycling vesicles in small CNS nerve terminals: implications for neural signaling and vesicular cycling.

The tiny nerve terminals of central synapses contain far fewer vesicles than preparations commonly used for analysis of neurosecretion. Photoconversion of vesicles rendered fluorescent with the dye FM1-43 directly identified vesicles capable of engaging in exo-endocytotic recycling following stimulated Ca(2+) entry. This recycling pool typically contained 30-45 vesicles, only a minority fraction (15-20% on average) of the total vesicle population. The smallness of the recycling pool would severely constrain rates of quantal neurotransmission if classical pathways were solely responsible for vesicle recycling. Fortunately, vesicles can undergo rapid retrieval and reuse in addition to conventional slow recycling, to the benefit of synaptic information flow and neuronal signaling.     

Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion

Exo-endocytotic turnover of synaptic vesicles (SVs) at synapses between hippocampal neurons in culture was examined by electron microscopy (EM). We carried out photoconversion (PC) of the fluorescent endocytotic marker FM 1-43 by using 3,3'-diaminobenzidine to convert the dye signal into an electron-dense product. Electron-dense products were located almost exclusively in SVs, whose densities were bimodally distributed in two sharply demarcated populations, PC-positive (PC+) and PC-negative (PC-). The median densities of these populations did not vary with the proportion of vesicles stained within a presynaptic terminal (bouton). The proportion of PC+ SVs remained constant across consecutive thin sections of single boutons, but varied greatly from one bouton to another, indicating marked heterogeneity in exo-endocytotic activity. Our experiments indicated that only a minority of SVs were stained in most boutons after stimuli known to cause complete turnover of the functional vesicular pool. A direct spatial correlation was found between FM 1-43 fluorescent spots seen with light microscopy and PC+ boutons by EM. The correlation was clearer in isolated boutons than in clusters of boutons. Photoconversion in combination with FM dyes allows clarification of important aspects of vesicular traffic in central nervous system nerve terminals.