Expression of the human dystrophin gene in mdx mouse skeletal muscles after ballistic transfection

OBJECTIVE: This study aimed to determine the prevalence of sensorineural hearing loss (SNHL) in 2-5-year-old survivors with neonatal respiratory failure due to congenital diaphragmatic hernia (CDH) with or without the need for extracorporeal membrane oxygenation (ECMO). STUDY DESIGN: The study design was a prospective, multicenter, longitudinal outcome study of consecutively surviving neonates admitted to a single tertiary intensive care unit. SETTING: The study was conducted at four audiologic departments affiliated with tertiary-level intensive care follow-up programs. PATIENTS: Thirty-seven surviving children receiving neonatal intensive care from February 1989 through January 1995 for neonatal respiratory failure due to CDH were studied. Excluded were 15 children with early death and I child lost to follow-up. INTERVENTION: The initial treatment depended on the severity of neonatal respiratory failure: ECMO-treated (n=31, 20 survivors) (death before ECMO initiation, 2) and non-ECMO treated (n=20, 17 survivors, another survivor lost to follow-up). MAIN OUTCOME MEASURE: Early childhood audiologic test results were measured. RESULTS: Sensorineural hearing loss was found in almost 60% of subjects: ECMO-treated, 12 (60%) of 20; non-ECMO-treated, 10 (59%) of 17. Of the 22 children with SNHL, 16 had mild- to-moderate low-frequency sloping to moderate-to-severe high-frequency loss. Of the remaining, six had severe-to-profound loss at 500 Hz and above. Seventeen children had normal responses to sound as newborns or in infancy. Five children were not tested. Documented progression was found in nine children. Twenty children currently are using amplification, and 2 have had cochlear implantation. CONCLUSIONS: Of children with CDH in this area presenting early with severe neonatal respiratory failure, SNHL developed in 60% by 2-5 years of life. Ongoing monitoring of the hearing status of children with CDH is imperative.     

Presynaptic modulation of cholecystokinin release by protein kinase C in the rat hippocampus

The role of protein kinase C (PKC) in modulating the release of the octapeptide cholecystokinin (CCK-8) was investigated in rat hippocampal nerve terminals (synaptosomes). The PKC-activating phorbol ester 4beta-phorbol 12,13-dibutyrate (beta-PDBu) dose dependently (5-5,000 nM) increased CCK-8 release in a strictly Ca2+dependent way. This effect was observed only when synaptosomes were stimulated with the K+(A) channel blocker 4-aminopyridine (4-AP; 1 mM) but not with KCl (10-30 mM). The PDBu-induced exocytosis of CCK-8 was completely blocked by the two selective PKC inhibitors chelerythrine and calphostin-C and was not mimicked by alpha-PDBu, an inactive phorbol ester. In addition, an analogue of the endogenous PKC activator diacylglycerol, oleoylacetylglycerol, dose dependently increased CCK-8 exocytosis. Beta-PDBu (50-100 nM) also stimulated the 4-AP-evoked Ca2+-dependent release of the classic transmitter GABA, which co-localizes with CCK-8 in hippocampal interneurons. As a possible physiological trigger for PKC activation, the role of the metabotropic glutamate receptor was investigated. However, the broad receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid did not stimulate, but instead inhibited, both the CCK-8 and the GABA exocytosis. In conclusion, presynaptic PKC may stimulate exocytosis of distinct types of co-localizing neurotransmitters via modulation of presynaptic K+ channels in rat hippocampus.     

TNF-R55-specific form of human tumor necrosis factor-alpha induces collagenase gene expression by human skin fibroblasts

Tumor necrosis factor-alpha (TNF-alpha) is a potent inhibitor of connective tissue formation. The cellular effects of TNF-alpha are mediated by two distinct cell-surface receptors, TNF-R55 and TNF-R75, both present on various types of cells, including fibroblasts. In this study we wanted to elucidate the role of TNF-R55 as a mediator of the connective tissue effects of TNF-alpha by using a mutant, TNF-R55-specific form of human TNF-alpha. This mutant TNF-alpha markedly induced collagenase and stromelysin-1 gene expression in dermal fibroblasts, the maximal activation (up to 42-fold) being 65%-89% of that noted with wild-type human TNF-alpha. In addition, TNF-R55-specific TNF-alpha suppressed type I collagen mRNA levels as potently as wild-type TNF-alpha (by 60%). The enhancement of collagenase gene expression by TNF-R55-specific TNF-alpha was augmented by simultaneous treatment of normal and scleroderma skin fibroblasts with interferon-gamma, indicating specific enhancement of TNF-R55 signaling pathway by interferon-gamma. These results show that stimulation of the TNF-R55 signaling pathway is sufficient for the inhibitory effects of TNF-alpha on extracellular matrix formation by dermal fibroblasts. It is conceivable that due to reduced systemic toxicity, TNF-R55-specific forms of human TNF-alpha may prove to be feasible in the therapy of fibrotic disorders.     

Anti-p53 antibodies in sera of workers occupationally exposed to vinyl chloride

BACKGROUND: The p53 tumor suppressor gene (also known as TP53) is often mutated in a wide variety of cancers, including angiosarcoma of the liver (ASL). Anti-p53 antibodies have been detected in the sera of patients with leukemia, childhood lymphoma, or cancers such as those of the breast, lung, colon, esophagus, and liver (hepatocellular carcinoma). PURPOSE: The objective of this study was to determine the prevalence and time of appearance of serum anti-p53 antibodies during the pathogenesis of ASL associated with occupational exposure to vinyl chloride. METHODS: Enzyme-linked immunoassay (EIA) was used to detect anti-p53 antibodies in 148 serum samples from 92 individuals occupationally exposed (in France or in Kentucky) to vinyl chloride; 15 of these individuals (six from France and nine from Kentucky) had ASL. A subset of coded EIA-positive and EIA-negative sera was further analyzed for anti-p53 antibodies by immunoblotting and immunoprecipitation. Nucleotide sequence analysis of exons 5-8 of the p53 gene was conducted on ASL DNA from six patients. We tested sera from 31 men who had no occupational exposure to vinyl chloride; they made up the control group. Statistical analyses were done using the Kruskal-Wallis chi-squared approximation and the Wilcoxon two-sample test for normal approximation. All P values result from two-sided tests. RESULTS: Fourteen serum samples (from nine individuals) were positive in the EIA. Five of the 15 individuals with ASL were positive for anti-p53 antibodies by EIA, immunoblotting, and immunoprecipitation: one individual at 11.3 and 10.8 years before diagnosis, another at 4 months before and shortly after diagnosis, and three when diagnosed or shortly thereafter. Four of the 77 vinyl chloride-exposed workers without diagnosed ASL were positive for anti-p53 antibodies; two of the four had symptoms related to vinyl chloride toxicity. Tumors from three of the six vinyl chloride-exposed workers from which sufficient DNA for analysis was obtained had A:T to T:A missense mutations of the p53 gene. Anti-p53 antibodies were detected in two of these individuals. Among the control group, two of 15 serum samples from 15 lung cancer patients and zero of 15 serum samples from control subjects without cancer had anti-p53 antibodies as substantially lower levels than the nine (10%) of 92 vinyl chloride-exposed workers who were positive for anti-p53 antibodies. CONCLUSIONS AND IMPLICATIONS: Serum anti-p53 antibodies can predate clinical diagnosis of certain tumors, such as ASL, and may be useful in identifying individuals at high cancer risk, such as workers with occupational exposure to vinyl chloride.     

The effects of different envelope patterns and uncertainty for the detection of a tone added to SAM complex tonal maskers

Thresholds for the detection of a tone added in-phase to the carrier of a fully modulated SAM tone were measured. In some conditions the signal was added to a single SAM tone, and in other conditions the signal was added to the sum of three or more SAM tones. Level equalization ensured that the addition of the tonal signal did not lead to increases in energy. When multiple SAM tone maskers were used, a small number of reproducible maskers were tested, each masker being composed of SAM tones with a variety of relative modulator phases. The maskers were either fixed across intervals and trials, roved across trials but fixed across intervals, or randomly chosen across both intervals and trials. The frequency separation between the signal-centered and off-frequency SAM tones was also varied. For small signal-centered/off-frequency SAM tone frequency separations, a separation ratio of 1.3, thresholds in the fixed condition depend on the relative modulator phases, and a simple mixture model reasonably predicted thresholds in the roving condition based on thresholds in the fixed condition for two of the three observers. For signal-centered/off-frequency SAM tone frequency separation factor of 1.68, effects of relative modulator phases were not obtained. Thresholds in the target-alone condition were generally superior to thresholds measured with the comodulated masker. Comodulated thresholds were better than target-alone thresholds only when level equalization was not used, and so the addition of the signal led to increases in level.     

Complement opsonization is required for presentation of immune complexes by resting peripheral blood B cells

Complement receptor 2 (CD21, CR2) is a B cell receptor for complement degradation products bound to Ag or immune complexes. The role of CD21 in mediating Ag presentation of soluble immune complexes by resting B cells was studied. Complement-coated immune complexes were formed by the incubation of influenza virus with serum from immune donors. These complexes bound to peripheral blood B cells in a complement-dependent manner. The binding required CD21 or, to a lesser extent, complement receptor 1 (CR1, CD35). B cells pulsed with immune complexes containing complement elicited a response from a panel of influenza-specific T cell clones, while those pulsed with immune complexes formed in the absence of complement did not. The expression of the early activation marker CD69 and the costimulatory molecule CD86 were not induced by CD21 ligation alone, suggesting that CD21-mediated Ag presentation occurs independently of B cell activation. Up-regulation of these markers required exposure to T cell factors elicited by the recognition of Ag derived from complement-containing immune complexes. These findings suggest that binding of Ag to CD21 enables Ag-nonspecific B cells to participate in the activation of Ag-specific T cells in a process that occurs independently of well-characterized B cell activation events.     

Seasonal changes in the activity of antioxidative defense in the kidneys of the euthermic ground squirrel (Citellus citellus)

The aim of this work was to determine the activity of the antioxidant enzymes: superoxide dismutase (EC 1.15.1.1; SOD), catalase (EC 1.11.1.6; CAT), glutathione peroxidase (EC 1.11.1.9; GSH-Px), glutathione-S-transferase (EC 2.5.1.18; GST), glutathione reductase (EC 1.6.4.2; GR) and the low molecular mass antioxidants: ascorbic acid (ASA) and vitamin E (vit E) in the kidney of ground squirrels during circannual changes. Keeping the ground squirrel at the temperature of thermic neutrality (30 degrees C) provides a stable euthermic state during the whole year and thus any change is due to the circannual rhythm. The highest specific activity of all examined antioxidative defense enzymes in the kidney was found in the spring, when ground squirrels are seasonally the most active. In the summer, lower specific activity of GSH-Px as well as of SOD and CAT were noted and, when expressed per g wet mass, only a decrease in GSH-Px activity was recorded. In the kidney of ground squirrels kept at 30 degrees C, the lowest specific activity of all examined enzymes was found during the winter and, when expressed per g wet mass, only the SOD activity was lower than in the spring and summer. Higher amounts of vitamins C and E were found in the ground squirrel kidneys in the summer. The results obtained in this work demonstrate that circannual regulation of metabolic activity, which is inherent to seasonal hibernators, is also expressed at the level of antioxidative defense in the kidneys.     

Lower gastrointestinal bleeding as the initial presenting symptom of renal cell carcinoma

Renal cell carcinoma is known as one of the "great mimics encountered in clinical medicine," along with syphilis and tuberculosis. It can present clinically as a wide range of symptoms, with a classic triad described as hematuria, pain, and a palpable abdominal mass. However, this triad is present only in <20% of patients with renal cell carcinoma. Gastrointestinal bleeding has been described in renal cell carcinoma, although mainly secondary to metastasis in the upper gastrointestinal tract, with few cases due to local invasion. Lower gastrointestinal bleeding as a presenting symptom of an invasive primary renal cell carcinoma has been described in only one patient in the literature. Our patient is the first in whom a colonoscopic biopsy was used as a successful diagnostic modality.     

The alloreactive T cell response against the class Ib molecule H2-M3 is specific for high affinity peptides

MHC class Ib molecule H2-M3 presents N-formylated peptides to CD8+ CTLs. Endogenous formylated peptides can come from the N-terminus of each of the 13 proteins encoded by the mitochondrial genome. In peptide competition assays, two of these peptides bind with high affinity, six bind with intermediate affinity, three bind with low affinity, and two do not bind measurably. Alloreactive CTLs from M3-specific, mixed lymphocyte cultures responded strongly against the two peptides with high affinity for M3, occasionally to peptides with intermediate affinity, and not at all to the rest. Long term lines and CTL clones reacted with only the high affinity peptides, demonstrating that alloreactive CTLs depend on specific peptides and that peptide affinity for class I correlates with alloantigenicity.