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Hormone control of autoantibodies to calf thymus nuclear extract (CTE) and DNA in MRL-lpr and MRL-+/+ mice.

Because hormonal influences on autoimmune disease in MRL-lpr and MRL-+/+ mice have not been defined completely, we examined animals which had been castrated and implanted with the opposite sex hormone. Antibodies directed at non-DNA antigens in a calf thymus nuclear extract (designated CTE) and specific anti-DNA antibodies were increased in estrogen-treated males, testosterone-treated females, and sham-operated female controls compared to sham-operated males. Analysis by sucrose gradient ultracentrifugation revealed that gonadal hormones exerted marked differences in the distribution and nature of circulating IgM anti-CTE antibodies. Although 19 S IgM was the predominant form of anti-CTE antibodies in experimental groups showing elevated anti-CTE responses, estrogen-treated male MRL-lpr mice expressed a large additional population of anti-CTE IgM antibody released by acid dissociation of apparently cryptic complexes. An unexpected additional finding was the presence of cryptic anti-CTE IgG (7 S) in all groups of MRL-lpr and MRL-+/+ mice, revealed only in sucrose gradient analysis under acid conditions. It is suggested that sex-related factors may account, in part, for apparent differences in levels of circulating autoantibodies observed in MRL mice by influencing the degree to which autoantibody populations exist in circulating complexes.

Animals

A control unit for maximal-rate continuous tissue expansion (CTE).

Subcutaneous or submuscular tissue expansion has become a major tool for the reconstructive plastic surgeon in the repair of difficult wounds and in breast reconstruction. This technique can provide additional or replacement tissue with good match in color, sensation, texture and adnexal characteristics. A major limitation of the technique is the time required for periodic tissue expansion to the required volume in the clinical setting, typically taking from a few weeks to several months. Conceptually, the maximum rate possible for safe clinical tissue expansion would result from continuous maintenance of tissue expander pressure just below capillary filling pressure (continuous tissue expansion, CTE). A control device with this capability would have important clinical and research applications. A self-contained portable control unit (patent pending) for CTE has been designed, fabricated, and testing initiated in a dog model. The 4-lb unit includes a 250-cc fluid reservoir, power pack, pressure transducer (Microswitch 160 PC), pump (Spalding 701A), feedback circuits and memory (2-lb power pack, 2-lb controller and reservoir). Preselected constant pressure (15-50 mmHg +/- 0.1 mmHg) can be maintained in a tissue expander with a maximal flow rate of 16 cc/hr for approximately 18 hours before change of the NiCad battery pack. A digital memory records pressure and volume infused and transfers this data to a Macintosh computer after completion of expansion. Verification of this prototype control unit is presently underway. Initial data suggests that the time required for complete filling of a subcutaneous tissue expander in a dog model can be reduced to approximately 72 hours. Several potential clinical applications and variations of this device are discussed.

Animals

Effects of chronic toluene exposure on central monoamine and peptide receptors and their interactions in the adult male rat.

The effects of chronic toluene exposure (CTE) (80 ppm, 6 h/day, 5 days/week, 3 months) were studied on neuropeptide and 5-hydroxytryptamine receptors, on protein phosphorylation levels and on catecholamine levels in various brain regions in the 15-month-old male rat. Behavioral parameters and serum levels of hypophyseal hormones and corticosterone were also analyzed. CTE selectively reduced [3H]neurotensin [( 3H]NT) binding in the basal layers of the orbital cortex. Instead, CTE increased the binding of [3H]etorphine in the nucleus accumbens and of [125I]vasoactive intestinal polypeptide [( 125I]VIP) in the area postrema and hypoglossal nucleus. Acute treatment with the irreversible monoamine receptor antagonist N-ethoxycarboxyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) increased the binding of [3H]NT in the orbital cortex in toluene exposed rats as compared with the reduced [3H]NT binding obtained in air exposed rats treated with EEDQ. Furthermore, the EEDQ induced increase in [125I]VIP binding in the area postrema and the hypoglossal nucleus was replaced by a reduced binding of [125I]VIP in EEDQ-treated CTE rats. CTE produced an overall increase in calcium-induced back phosphorylation and an overall decrease in cyclic adenosine monophosphate-induced back phosphorylation in the frontoparietal cortex. Noradrenaline stores tended to be reduced within various hypothalamic subnuclei and the serum prolactin levels were increased following CTE. However, no marked effects of CTE were seen on the behavioral parameters. In conclusion, the regional selectivity of CTE in disturbing [3H]NT and [125I]VIP binding may be due to the demonstrated vulnerability of monoamine-neuropeptide interactions to toluene.

Animals

Effect of hormones on growth and function of cultured canine tracheal epithelial cells.

Insulin (INS), endothelial cell growth supplement (ECGS), transferrin (TF), cholera toxin (CT), hydrocortisone (HC), triiodothyronine (T3), and epidermal growth factor (EGF) were systematically examined for their effects on proliferation, ion transport activity, and morphological differentiation of canine tracheal epithelial (CTE) cells in culture. INS, ECGS, TF, and CT increase proliferation of CTE cells cultured on plastic Petri dishes but exhibit no acute effect on the bioelectric activity of freshly excised CTE. CT increases amiloride-insensitive transepithelial ion transport across both freshly excised canine trachea and CTE cultures. EGF has no effect on proliferation of CTE cells on plastic but induces hyperplasia of CTE cells on collagen matrices. EGF does not alter basal transepithelial ion transport across cultures but increases cellular responsiveness to beta-adrenergic stimulation. HC and T3 have no effect on proliferation or transepithelial bioelectric properties of CTE cells but improve morphological differentiation yielding cultures of complex epithelia containing cuboidal ciliated and nonciliated cells. These results demonstrate that growth and function of cultured CTE cells are affected by specific growth factors.

Amiloride

Aerobic dehalogenation of halothane showing different substrate dependency from anaerobic dehalogenation in liver microsomes of guinea pig.

The formation of trifluoroacetic acid (TFAA) from halothane under aerobic conditions and that of chlorotrifluoroethane (CTE) and chlorodifluoroethylene (CDE) from halothane under anaerobic conditions were studied using guinea pig liver microsomes. The formation of TFAA was inhibited by specific inhibitors of cytochrome P450 (P450), such as carbon monoxide and metyrapone and was dependent upon P450 contents. The maximum activity of the TFAA formation was obtained at pH 6.0. On the other hand, the maximum activity to form CTE and CDE was obtained at pH 7.4. The formation of TFAA reached a plateau at a halothane concentration above 0.17 mM, but the rate of formation of CDE and CTE was dependent upon a halothane concentration up to 1.5 mM. The values of apparent Michaelis-Menten constant (Km) and maximum velocity (Vmax) for TFAA formation were 0.067 mM and 0.349 nmol/nmol P450/min respectively, those for CDE formation were 0.983 mM and 0.326 nmol/nmol P450/min respectively, and those for CTE formation were 1.71 mM and 0.752 nmol/nmol P450/min respectively. These results showed clearly that the formation of TFAA, CDE and CTE was catalyzed by the P450 system in guinea pig liver microsomes. Under optimal conditions, saturation was observed in the formation of TFAA from halothane at a halothane concentration above 0.17 mM but the formation of CDE and CTE was not saturated at this concentration, and the value of apparent Km for TFAA formation was lower than those for CDE and CTE formation.

Aerobiosis

[Localization of fluorescent analogs of cholesterol and its esters in lipid models of membranes and lipoproteins].

The localization of the fluorescent cholesterol analogue--delta 5,7,9(11)-cholestatrien-3 beta-ol (B-CTE) and its methyl (M-CTE) and stearoyl (St-CTE) esters in model lipid particles were investigated by the radiationless energy transfer. It is shown that 67-100% of B-CTE molecules localize in the phospholipid monolayer of particles containing phosphatidylcholine and triolein (1:2 w/w). The replacement of a hydrogen atom in the hydroxyl by methyl resulted in immersion of 2/3 of the M-CTE molecules to the triolein core. This fact confirmed distribution of the fluorescent probe molecules in the whole volume of lipid particles. St-CTE was practically completely localized in the core of lipid particles. The results obtained evidence for the important role of 3-OH group in keeping the B-CTE molecules in the phospholipid monolayer of the investigated particles.

Cholestenes

In vivo biological effect of allogeneic cultured thymic epithelium on thymus-dependent immunity in athymic nude rats.

We have extended our previous study of induction of T-lymphocyte immunocompetence in athymic nude rats by cultured epithelium (CTE) of syngeneic origin to that by CTE of allogeneic origin. Immune responsiveness (IgG-class antibody and delayed-type hypersensitivity) after ovalbumin immunization is detectable by 4-6 weeks after transplantation. However, the antibody appears at a slower rate when compared with heterozygous immunocompetent littermates. Seven weeks after transplantation phytohaemagglutinin responsiveness of spleen cells is detectable, and in T-dependent areas of lymphoid organs lymphocytes with helper and non-helper T-cell phenotype are present, but at lower levels than those in heterozygous immunocompetent littermates. Levels comparable to that of immunocompetent rats are reached about 20 weeks after transplantation. Since CTE contains thymocytes, control experiments consisted of transplantation with high numbers of allogeneic freshly isolated thymocytes in athymic nude rats. These animals showed IgG-class antibody formation after ovalbumin immunization, but at lower levels than CTE-treated rats, and were almost negative in T-cell immunocompetence assessed in the other assays. We conclude that CTE of allogeneic origin induces T-cell immunocompetence in athymic nude rats to the level of heterozygous immunocompetent littermates. This study adds to the rationale of CTE transplantation applied in treatment of thymic dysfunction.

Animals

A recombinant 70K protein ELISA. Screening for antibodies against U1snRNP proteins in human sera.

Antibodies to uridylic acid rich small nuclear ribonucleoprotein particles (UsnRNP) are mainly detected in patients with systemic lupus erythematosus (SLE) or mixed connective tissue disease (MCTD). Particularly those directed against epitopes of the 70K protein of U1snRNP serve as important markers for the diagnosis of MCTD. To establish an ELISA for determination of anti-70K protein antibodies in patients' sera a 1239 bp long cDNA insert coding for the epitopes of the 70K protein was ligated into a fusion expression vector. The bacterially expressed fusion protein was purified by chromatography on DEAE cellulose. Microtiter plates were coated with the fusion protein as well as with partially purified calf thymus extract (CTE) containing all natural UsnRNP antigens and RNase digested calf thymus extract (CTERNase) in which the natural 70K antigen was destroyed by the nuclease treatment. 10,888 sera of patients with suspected or overt rheumatic disease were analyzed for antibodies against these antigens simultaneously. Antibodies against CTE or CTERNase were not detected in 9123 sera, none of these showed reactivity with the 70K protein indicating a high degree of specificity of the assay. Positive results in each the 70K protein, CTE as well as the CTERNase ELISAs were obtained with 474 sera. 319 sera were only positive with CTE and 70K protein. Of these 793 anti-70K protein ELISA positive sera, 79% could be confirmed by immunoblot. Of 967 sera reacting with CTE and CTERNase but not with the recombinant 70K protein, 31% contained antibodies against various other UsnRNP proteins as shown by immunoblotting. 2.4% of these sera revealed also antibodies against the 70K protein. The use of the recombinant 70K protein as antigen meets the criterion for a simple and specific assay to detect anti-U1snRNP antibodies. Nevertheless, the sole use of this recombinant protein for anti-U1snRNP antibody screening may not be appropriate, because antibodies against other frequently occurring U1snRNP proteins (A, C) cannot be detected with this test. Therefore it should be used together with a natural UsnRNP antigen until further studies in patients with well established diagnoses will show whether natural antigens may be omitted.

Antibody Specificity

Interleukin 2 responsive T cell clones from rheumatoid and normal subjects: proliferative responses to connective tissue elements.

In vivo-activated interleukin 2 responsive T cell clones were generated from peripheral blood (PB) and synovial fluid (SF) of rheumatoid arthritis (RA) patients and from normal control PB. The specificity of these clones was assessed by measuring proliferation induced by the connective tissue elements (CTE) collagen types I and II, native and denatured, proteoglycans, and irrelevant control antigens. The cloned T cells from RA patients but not from normal subjects responded in vitro with proliferation to all CTE but not to control antigens purified protein derivative, ovalbumin, or lysozyme. Proliferation occurred in the presence and absence of accessory cells (AC), but the responses were consistently higher in the presence of AC. Antibodies to HLA-DR abrogated the proliferative response to CTE suggesting that DR expression was necessary for the induction of proliferation. These findings demonstrate the existence of clonable T cells responsive to CTE in PB and SF of RA patients. Expression of reactivity to CTE may contribute to the chronicity of the inflammation in RA.

Arthritis, Rheumatoid

Modulation of the reductive metabolism of halothane by microsomal cytochrome b5 in rat liver.

To study the modulation of the reductive metabolism of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) by microsomal cytochrome b5, formation of 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), major reduced metabolites of halothane, was analyzed in vivo and in vitro. Rats were pretreated with both malotilate (diisopropyl-1,3-dithiol-2-ylidenemalonate) and sodium phenobarbital (malotilate-treated rats) or only with sodium phenobarbital (control rats). The microsomes of malotilate-treated rats had significantly more cytochrome b5 than the controls, whereas the cytochrome P-450 content was not different between the two groups. At the end of 2-h exposure to 1% halothane in 14% oxygen, the ratio of CDE to CTE in arterial blood was significantly higher in malotilate-treated rats than in the controls. Under anaerobic conditions, the formation of CDE and the ratio of CDE to CTE were significantly greater in microsomal preparations of malotilate-treated rats than those of the controls. In a reconstituted system containing cytochrome P-450PB purified from rabbit liver, addition of cytochrome b5 to the system enhanced the formation of CDE and increased the ratio of CDE to CTE. These results suggested that cytochrome b5 enhances the formation ratio of CDE to CTE by stimulating the supply of a second electron to cytochrome P-450, which might reduce radical reactions in the reductive metabolism of halothane.

Anaerobiosis

Quantitative analysis of volatile halothane metabolites in biological tissues by gas chromatography.

A simple and sensitive gas chromatographic method for the determination of 2-chloro-1, 1-difluoroethylene (CDE) and 2-chloro-1,1,1-trifluoroethane (CTE), two highly volatile metabolites of halothane, in blood, liver and isolated hepatic microsomes is described. The entire head-space in equilibrium with a known volume or weight of the sample is injected into the gas chromatograph equipped with a flame ionization detector. Quantification is accomplished with standards prepared by fortifying blank samples with known concentrations of CDE and CTE which are treated under the same conditions as the samples. Detection limits for CDE and CTE were 2 pmole/ml in blood and 10 pmole/g in liver and the mean relative standard deviations are no greater than +/- 6% except for CTE in hepatic microsomes (+/- 9%). A preliminary study of blood CDE and CTE levels in humans anesthetized with halothane is reported.

Chromatography, Gas

A novel form of ectopic human chorionic gonadotrophin beta-subunit in the serum of a woman with epidermoid cancer.

A novel form of free human chorionic gonadotrophin beta-subunit (hCG beta) was found in serum from ElBre, a woman with epidermoid carcinoma of unknown origin. ElBre hCG beta was larger than standard (pregnancy urine) hCG beta when analysed by gel chromatography (apparent molecular weight 54 000 vs 44 000). This size difference appeared to be due to a larger carboxyterminal extension (CTE) of ElBre hCG beta since thermolysin cleavage of the CTE from standard hCG beta and Elbre hCG beta yielded core products of the same size. Oligosaccharides, O-linked to serine or threonine, were present in ElBre hCG beta, presumably on its CTE as judged by the complete binding of desialylated ElBre hCG beta to immobilized peanut agglutinin (this lectin is specific for terminal galactose linked beta 1----3 to N-acetylgalactosamine, a disaccharide exposed after desialylation of the O-linked oligosaccharides of standard hCG beta). ElBre hCG beta, however, was incompletely recognized by antisera specific for the CTE of standard hCG beta, especially the carbohydrate-sensitive antiserum R141. The O-linked oligosaccharides of standard hCG beta are heterogeneous in size; 13% are of the largest (hexasaccharide) form. In contrast, over 50% of the O-linked oligosaccharides in hCG beta from the JAr choriocarcinoma cell line are hexasaccharides. Like desialylated ElBre hCG beta, desialylated JAr hCG beta bound completely to peanut agglutinin, but was incompletely recognized by antisera to the hCG beta-CTE. Furthermore, JAr hCG beta was intermediate in size between standard hCG beta and ElBre hCG beta when analysed by gel chromatography (apparent molecular weight 49 000).(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Squamous Cell

Formation of bacterial mutagens from the reaction of chewing tobacco with nitrite.

Using the Salmonella/microsome assay system, the mutagenicity of chewing tobacco extracts (CTE) treated with and without sodium nitrite under acidic conditions was examined. Mutagenic activity was found only for nitrite-treated CTE in both tester strains, TA98 and TA100, and was independent of metabolic activation. Formation of mutagenic substances from CTE by nitrite was dependent on acidic pHs (the highest at pH 2) and could be inhibited by ascorbate. The mutagenic potency of CTE plus nitrite was proportional to the content of nitroso compounds generated in the reaction mixture, indicating that the nitrosation process was involved. The possible in vivo nitrosation and the potential health effect are discussed.

Animals

Isolation and characterization of chicken thymic electrolectin.

We have detected the presence of a beta-D-galactoside-binding lectin (electrolectin) in extracts of the thymus of adult chickens. This lectin was purified by affinity chromatography on a lactosyl-Sepharose column to yield 1.4 mg of pure protein from 230 g of thymus. The chicken thymic electrolectin (CTE) has an Mr of 15 300 when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and of 30 000 when analysed by gel filtration. The amino acid composition of CTE is similar to that of other electrolectins purified from human and rat lung. CTE cross-reacts immunologically, but is not identical, with electrolectins from electric-eel electric organ and from chick-embryo pectoral muscle. CTE agglutinates chicken thymocytes but does not appear to promote their mitosis.

Amino Acids

Active multiple sclerosis. Enhanced computerized tomographic imaging of lesions and the effect of corticosteroids.

Computerized axial transmission tomography (CT) of the brain is useful for imaging lesions in multiple sclerosis (MS). Active demyelination may be demonstrated with CT contrast enhancement (CTE) as regions of increased x-ray density. We report a series of patients with active MS who typify these changes. Corticosteroid therapy reduces the intensity of this phenomenon presumably by reestablishing the integrity of the blood-brain barrier; if corticosteroid therapy is instituted prior to the CT contrast study, the focal enhancement may be obscured. A transient vascular permeability defect is the basis for CTE during the acute exacerbation in MS. The possibility of MS must be kept in mind when one or more foci of increased density occur in the absence of mass effect during CTE. Appreciation of these features may prevent misdiagnosis.

Adult

Evidence for secretion of high molecular weight mucins by canine tracheal epithelial cells in primary culture: effects of select secretagogues in mucin secretion.

The purpose of this investigation was to provide evidence for the secretion of high molecular weight mucins, CTM-A and CTM-B, in primary culture of canine tracheal epithelial (CTE) cells. The cells were isolated from tracheas of mongrel dogs by pronase treatment. Primary cultures of the epithelial cells were established using ICN collagen inserts in Dulbecco's modified Eagle's/F12 medium supplemented with growth factors and could be maintained for up to 23 days. The evidence for the mucin secretion in culture medium and their localization in the cells was established by a) positive immunocytochemical staining using specific antibodies developed against purified native as well as deglycosylated CTM-A and CTM-B; b) incorporation of labeled amino acids, followed by electrophoresis and autoradiography detection of glycoconjugates purified from the culture medium; c) comparison of the amino acid compositions of mucin purified from canine tracheal pouch secretions and that purified from the culture medium; and d) Western blot analyses using specific polyclonal antibodies directed against deglycosylated CTM-A and CTM-B. Immunoaffinity purified secreted labeled glycoconjugates were resistant to hyaluronidase treatment. The effects of cyclic AMP (1 x 10(-5) M), dibutyryl cyclic AMP (1 x 10(-5) M), 8-bromocyclic AMP (1 x 10(-5) M), and prostaglandin E1 (1 x 10(-6) M) on mucin secretion by CTE cells were also investigated. Secretion of mucins by CTE cells in culture was considerably more enhanced by 8-bromocyclic AMP than that observed for other secretagogues used in this study.

Alprostadil

Reductive metabolism of halothane by purified cytochrome P-450.

The reductive metabolism of halothane was determined using purified RLM2, PBRLM4 and PBRLM5 forms of rat liver microsomal cytochrome P-450. The metabolites, 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), were determined. All three forms of cytochrome P-450 produced CTE with relatively small differences in its production among the various forms. There were major differences, however, in the production of CDE, with PBRLM5 being the most active. PBRLM5 was also the only form to show the development of a complex between halothane and cytochrome P-450. This complex absorbed light maximally at 470 nm. The complex formation and the production of CDE by PBRLM5 were stimulated by the addition of cytochrome b5. Cytochrome b5 had no effect on CDE production by PBRLM4 and inhibited the production of both CTE and CDE by RLM2. These results show that the two-electron reduction of halothane by cytochrome P-450 was catalyzed by the PBRLM5 form and that cytochrome b5 stimulated the transfer of the second electron to halothane through PBRLM5, but not RLM2 or PBRLM4.

Animals