Phenotypic and functional differences between human dendritic cells derived in vitro from hematopoietic progenitors and from monocytes/macrophages

The kinetics of inactivation of cytochrome P450 2B1, the major phenobarbital inducible rat hepatic P450, by N-benzyl-1-aminobenzotriazole (BBT) were characterized. Purified, reconstituted P450 2B1 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylase activity was inhibited by BBT in a mechanism-based manner. The loss of O-deethylase activity followed pseudo-first-order kinetics and was NADPH and BBT dependent. After a 5 min incubation, greater than 90% of the 2B1 activity was lost, whereas more than 70% of the ability of the reduced enzyme to bind CO was maintained. Inclusion of 10 mM glutathione in the inactivation reaction lowered the rate of inactivation (k(inactivation)) and increased the partition ratio without significantly affecting the inactivator concentration required for half-maximal inactivation (K(I)). The maximal rate constant for inactivation at 23 degrees C was 0.24 min(-1) without and 0.15 min(-1) with glutathione. The apparent K(I) was 2 microM in both cases. The extrapolated partition ratios were 4 and 9 without and with 10 mM glutathione, respectively. Consistent with mechanism-based inactivation, the loss of 7-EFC O-deethylase activity was irreversible, was not due to product inhibition, was saturable, and could be slowed by including increasing concentrations of competing substrate. However, the inactivated P450 2B1 was still able to metabolize substrate if iodosobenzene was used as an alternate oxidant. Inactivation of 2B1 with either N-[14C]-7-benzyl-1-aminobenzotriazole (BBT) or N-benzyl-1-amino-[14C]-2,3-benzotriazole resulted in the incorporation of covalent radiolabel into the apoprotein. The stoichiometry of labeled metabolite adduct to protein was approximately 0.4:1 in both cases. Identification of metabolites revealed the formation of 1-aminobenzotriazole, benzotriazole, benzaldehyde, and a new metabolite (27) during catalysis of BBT by P450 2B1. Together, these data suggest that P450 2B1 could be inactivated and labeled by more than one metabolite.     

Effect of the use of bovine somatotropin on culling practices in thirty-two dairy herds in Indiana, Michigan, and Ohio

Data from 5468 Holstein dairy cows in 32 herds were used to determine the effect of the use of bovine somatotropin (bST) on culling practices over a 13-mo period. After an initial herd inventory, monthly information regarding cow entry and exit from the herds was obtained by seven participating veterinarians. Culling was coded by farmers for the following reasons: low production, reproduction, somatic cell count, mastitis, sickness, dairy purposes, lameness, death, and other. In the control herds, cows were not treated with bST during the trial. Adopter herds were defined as herds that utilized supplemental bST for > or = 25% of the cow-days during the trial. Mean herd use of bST in adopter herds was 38.6%. No difference in the number of cows culled per cow-day at risk was detected between control and adopter herds (0.09 and 0.11%, respectively). Amount of in-herd use of bST was unrelated to culling. No significant differences were determined between adopter and control herds in the percentage of cows that were culled for any of the nine possible culling reasons. The results of this study suggest that culling patterns in herds that use bST are unaffected for at least the first year after adoption.     

Central nervous system delivery of interleukin 4 by a nonreplicative herpes simplex type 1 viral vector ameliorates autoimmune demyelination

Multiple sclerosis (MS) is a T cell-mediated organ-specific inflammatory disease leading to central nervous system (CNS) demyelination. On the basis of results obtained in experimental autoimmune encephalomyelitis (EAE) models, MS treatment by administration of antiinflammatory cytokines such as interleukin 4 (IL-4) is promising but is hampered by the limited access of the cytokines to the CNS and by the pleiotropic effects of systemically administered cytokines. We established a cytokine delivery system within the CNS using non-replicative herpes simplex type 1 (HSV-1) viral vectors engineered with cytokine genes. These vectors injected into the cisterna magna (i.c.) of mice diffuse in all ventricular and subarachnoid spaces and infect with high efficiency the ependymal and leptomeningeal cell layers surrounding these areas, without obvious toxic effects. Heterologous genes contained in the vectors are efficiently transcribed in infected ependymal cells, leading to the production of high amounts of the coded proteins. For example, 4.5 ng of interferon gamma (IFN-gamma) per milliliter is secreted into the cerebrospinal fluid (CSF) up to day 28 postinjection (p.i.) and reaches the CNS parenchyma in bioactive form, as demonstrated by upregulation of MHC class I expression on CNS-resident cells. We then exploited the therapeutic potential of the vectors in EAE mice. An HSV-1-derived vector containing the IL-4 gene was injected i.c. in Biozzi AB/H mice at the time of EAE induction. We found the following in treated mice: (1) delayed EAE onset, (2) a significant decrease in clinical score, (3) a significant decrease in perivascular inflammatory infiltrates and in the number of macrophages infiltrating the CNS parenchyma and the submeningeal spaces, and (4) a reduction in demyelinated areas and axonal loss. Peripheral T cells from IL-4-treated mice were not affected either in their antigen-specific proliferative response or in cytokine secretion pattern. Our results indicate that CNS cytokine delivery with HSV-1 vectors is feasible and might represent an approach for the treatment of demyelinating diseases. Advantages of this approach over systemic cytokine administration are the high cytokine level reached in the CNS, the absence of effects on the peripheral immune system, and the long-lasting cytokine production in the CNS after a single vector administration.     

Serum dioxin levels in Seveso, Italy, population in 1976

On July 10, 1976, an explosion at a chemical plant near Seveso, Italy, released a mixture of chemicals, including 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,4,5-trichlorophenol. As a result, several thousand people in the Seveso area may have been exposed to those chemicals. At that time, human exposure assessment was based primarily on soil levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Medical examinations of this potentially exposed population and control subjects were begun in 1976 and in some cases continued until 1985. In 1988, we began assessing human exposure in this population by measuring 2,3,7,8-tetrachlorodibenzo-p-dioxin in small volumes of serum specimens remaining from the medical examinations. As expected, we found that the median serum dioxin levels were highest among people who lived closest to the explosion and were progressively lower among groups living farther away. These measurements have allowed us to assess exposure more accurately among individuals in this population and to relate exposure to various health effects. We found that some individuals in the exposed population had among the highest serum dioxin levels ever reported, yet chloracne was the only unequivocal effect found; cancer risks are still being investigated. We also found that other individuals with as high or higher serum dioxin levels did not develop chloracne. We also found that the serum half-life of dioxin in this population was 7-8 years, which agrees with other findings although we do report some differences in the serum half-life of TCDD for women and children. We also observed an increase in the percentage of female newborns to parents who resided in Zone A at the time of the explosion, and we also report on the 1976 serum dioxin levels in people who later developed cancer.     

Differential roles of p300 and PCAF acetyltransferases in muscle differentiation

To determine the possibility of discriminating multi-sources in the brain by 3D vector magnetic field measurement of a magnetoencephalogram (MEG), measurements were made of magnetic fields produced by two current dipoles implanted in a spherical head model. The 3D vector magnetic field measurements were made by using a 3D second-order gradiometer connected to three rf-SQUIDs, which can detect magnetic field components perpendicular to and tangential to the scalp. The MEG distribution measuring the magnetic field perpendicular to the scalp was not helpful in estimating the location and number of sources because of the lack of a dipole pattern. By referring to the MEG distribution measuring the magnetic field distribution tangential to the scalp, however, two current sources could be clearly discriminated in a spherical head model. It was found that this MEG distribution measuring tangential to the scalp could provide information on new constraint conditions for the calculation of inverse problems with multi-sources. These results were also confirmed by measurement of the mixed somatosensory evoked fields elicited by simultaneous electric stimulation to the median nerve and the thumb.     

Lipoma and liposarcoma: evaluation using CT and MR imaging

The multi-subunit NADH-ubiquinone oxidoreductase (complex I) is the first enzyme complex in the electron transport chain of mitochondria. A small number of NADH-ubiquinone oxidoreductase subunits are the products of mitochondrial genes (subunits 1-7), while the remainder are nuclear encoded and imported from the cytoplasm. We have isolated and sequenced five subunits of the human complex I from a human heart lambda ZAP Express cDNA library. Comparison of the deduced amino acid sequences of the human subunits with the corresponding bovine sequences revealed greater than 80% amino acid identity. The high degree of similarity between human and bovine sequences suggests functional conservation of these subunits in the complex I. In silico Northern analysis revealed that two of the subunits were expressed ubiquitously while the remainder may have more restricted patterns of expression.     

Fate of microinjected sperm components in the mouse oocyte and embryo

Combining HB vaccine with routine paediatric vaccines has been recognized as the best means of universal vaccination against hepatitis B. Our objective was to evaluate the long-term antibody persistence of such a combined vaccine in an area of high hepatitis B endemicity. We have shown that a DTPw-HB vaccine was safe and immunogenic when given as a booster dose at 18 months of age. One month after the booster dose of DTPw-HB vaccine, at least 97.8% of subjects had seroprotective anti-HBsAg levels, and 1 year later at least 93.9% of these subjects remained seroprotected against HBsAg. Immune responses to the DTPw components were similar or greater than those of the commercial DTPw vaccine given to the control group. This DTPw-HB vaccine, which showed good long-term anti-HBsAg antibody persistence, could advantageously replace separate DTPw and HB vaccines in areas of high hepatitis B endemicity in terms of clinical, economic and strategic benefits.     

Variables controlling contrast generation in a urinary bladder model

An ultrasound system has been developed to generate microbubbles in vivo for use as ultrasound contrast agent. Possible application include diagnosis of reflux in the urinary tract. In experiments designed to elucidate the contrast microbubble generation process, acoustic bursts (at 1.8 MHz, 125 ms) were propagated through a latex rubber balloon, modeled after a rabbit urinary bladder, containing fluids of various air and carbon dioxide saturations and concentrations of cavitation nuclei (0.198-micron-diam polystyrene particles). The peak rarefactional pressure threshold for contrast microbubble generation, as visualized with a diagnostic ultrasound system, decreased approximately a factor of 2 for increasing particle concentration from 10(8) to 10(10) particles/cc, with the lowest threshold of 5.24 MPa. For samples with gas saturations below 50% and 10(10) particles/cc, the average thresholds were at least twice as high as those of more saturated fluids (with mean threshold for saturated fluids of 6.45 MPa), and samples containing CO2 had considerably lower thresholds than respective under-saturations in air. At a fixed pressure amplitude, echogenicity tended to increase with both increasing particle concentration and gas saturation; this was more favorable for samples containing CO2. Even in a restricted-nuclei environment such as the urinary bladder, generation of vaporous cavitation should be possible; however, subsequently, abundant gas is needed to grow vaporous bubbles to persistent and imageable sizes, to assist in the diagnosis of urinary reflux.     

Agonist-induced endocytosis and recycling of the gonadotropin-releasing hormone receptor: effect of beta-arrestin on internalization kinetics

This study examined the dynamics of endocytotic and recycling events associated with the GnRH receptor, a unique G protein-coupled receptor (GPCR) without the intracellular carboxyl-terminal tail, after agonist stimulation, and investigated the role of beta-arrestin in this process. Subcellular location of fluorescently labeled epitope-tagged GnRH receptors stably expressed in HEK 293 cells was monitored by confocal microscopy, and the receptor/ligand internalization process was quantified using radioligand binding and ELISA. Agonist stimulation resulted in reversible receptor redistribution from the plasma membrane into the cytoplasmic compartment, and colocalization of internalized GnRH receptors with transferrin receptors was observed. Internalization experiments for the GnRH receptor and another GPCR possessing a carboxy-terminal tail, the TRH receptor, showed that the rate of internalization for the GnRH receptor was much slower than for the TRH receptor when expressed in both HEK 293 and COS-7 cells. TRH receptor internalization could be substantially increased by coexpression with beta-arrestin in COS-7 cells, while GnRH receptor internalization was not affected by coexpression with beta-arrestin in either cell type. Coexpression of the GnRH receptor with the dominant negative beta-arrestin (319-418) mutant did not affect its ability to internalize, and activated GnRH receptors did not induce time-dependent redistribution of beta-arrestin/green fluorescent protein to the plasma membrane. However, the beta-arrestin mutant impaired the internalization of the TRH receptor, and activated TRH receptors induced the beta-arrestin/green fluorescent protein translocation. This study demonstrates that, despite having no intracellular carboxy-terminal tail, the GnRH receptor undergoes agonist-stimulated internalization displaying distinctive characteristics described for other GPCRs that internalize via a clathrin-dependent mechanism and recycle through an acidified endosomal compartment. However, our data indicate that the GnRH receptor may utilize a beta-arrestin-independent endocytotic pathway.     

Treatment of subglottic laryngitis (pseudocroup): steroids instead of steam]

Traditionally, steaming with warm moist air was recommended for the treatment of subglottic laryngitis (pseudocroup). However, no favourable effect has ever been demonstrated. Consequently, steaming is no longer to be advised. Systemic corticosteroids, already of proven effectiveness in severe croup, were shown to be also effective when administered in a single oral dose in moderately severe disease. Besides, in various studies, nebulisation of budesonide (2000 micrograms) with a jet nebuliser had a good effect on the clinical course of croup. However, dose-effect studies are still lacking. A single dose of corticosteroids, either systemic or inhaled via a jet nebuliser, should be the first line therapy in moderate and severe croup syndrome. In milder cases no specific treatment is needed as the disease is self-limiting.