Regional airflow obstruction after bronchoconstriction and subsequent bronchodilation in subjects without pulmonary disease.

Some subjects with asthma have ventilation defects that are resistant to bronchodilator therapy, and it is thought that these resistant defects may be due to ongoing inflammation or chronic airway remodeling. However, it is unclear whether regional obstruction due to bronchospasm alone persists after bronchodilator therapy. To investigate this, six young, healthy subjects, in whom inflammation and remodeling were assumed to be absent, were bronchoconstricted with a PC20 [the concentration of methacholine that elicits a 20% drop in forced expiratory volume in 1 s (FEV1)] dose of methacholine and subsequently bronchodilated with a standard dose of albuterol on three separate occasions. Specific ventilation imaging, a proton MRI technique, was used to spatially map specific ventilation across 80% of each subject's right lung in each condition. The ratio between regional specific ventilation at baseline and after intervention was used to classify areas that had constricted. After albuterol rescue from methacholine bronchoconstriction, 12% (SD 9) of the lung was classified as constricted. Of the 12% of lung units that were classified as constricted after albuterol, approximately half [7% (SD 7)] had constricted after methacholine and failed to recover, whereas half [6% (SD 4)] had remained open after methacholine but became constricted after albuterol. The incomplete regional recovery was not reflected in the subjects' FEV1 measurements, which did not decrease from baseline (P = 0.97), nor was it detectable as an increase in specific ventilation heterogeneity (P = 0.78).NEW & NOTEWORTHY In normal subjects bronchoconstricted with methacholine and subsequently treated with albuterol, not all regions of the healthy lung returned to their prebronchoconstricted specific ventilation after albuterol, despite full recovery of integrative lung indexes (forced expiratory volume in 1 s and specific ventilation heterogeneity). The regions that remained bronchoconstricted following albuterol were those with the highest specific ventilation at baseline, which suggests that they may have received the highest methacholine dose.

Spatial persistence of reduced specific ventilation following methacholine challenge in the healthy human lung.

Specific ventilation imaging was used to identify regions of the healthy lung (6 supine subjects, ages 21-41 yr, 3 men) that experienced a fall in specific ventilation following inhalation of methacholine. This test was repeated 1 wk later and 3 mo later to test for spatial recurrence. Our data showed that 53% confidence interval (CI; 46%, 59%) of volume elements that constricted during one methacholine challenge did so again in another and that this quantity did not vary with time; 46% CI (28%, 64%) recurred 1 wk later, and 56% CI (51%, 61%) recurred 3 mo later. Previous constriction was a strong predictor for future constriction. Volume elements that constricted during one challenge were 7.7 CI (5.2, 10.2) times more likely than nonconstricted elements to constrict in a second challenge, regardless of whether the second episode was 1 wk [7.7 CI (2.9, 12.4)] or 3 mo [7.7 CI (4.6, 10.8)] later. Furthermore, posterior lung elements were more likely to constrict following methacholine than anterior lung elements (volume fraction 0.43 ± 0.22 posterior vs. 0.10 ± 0.03 anterior; P = 0.005), and basal elements that constricted were more likely than their apical counterparts to do so persistently through all three trials (volume fraction 0.14 ± 0.04 basal vs. 0.04 ± 0.04 apical; P = 0.003). Taken together, this evidence suggests a physiological predisposition toward constriction in some lung elements, especially those located in the posterior and basal lung when the subject is supine. NEW & NOTEWORTHY The spatial pattern of bronchoconstriction following methacholine is persistent over time in healthy individuals, in whom chronic inflammation and airway remodeling are assumed to be absent. This suggests that regional lung inflation and airway structure may play dominant roles in determining the spatial pattern of methacholine bronchoconstriction.

Profiling solute carrier transporters in the human blood-brain barrier.

The neuroprotective function of the blood-brain barrier (BBB) presents a major challenge for drug delivery to the central nervous system (CNS). Critical to this function, BBB membrane transporters include the ATP-binding cassette (ABC) transporters, which limit drug penetration across the BBB, and the less-well-studied solute carrier (SLC) transporters. In this work, expression profiling of 359 SLC transporters, comparative expression analysis with kidney and liver, and immunoassays in brain microvessels (BMVs) identified previously unknown transporters at the human BBB.

A giant protease with potential to substitute for some functions of the proteasome.

An alanyl-alanyl-phenylalanyl-7-amino-4-methylcoumarin-hydrolyzing protease particle copurifying with 26S proteasomes was isolated and identified as tripeptidyl peptidase II (TPPII), a cytosolic subtilisin-like peptidase of unknown function. The particle is larger than the 26S proteasome and has a rod-shaped, dynamic supramolecular structure. TPPII exhibits enhanced activity in proteasome inhibitor-adapted cells and degrades polypeptides by exo- as well as predominantly trypsin-like endoproteolytic cleavage. TPPII may thus participate in extralysosomal polypeptide degradation and may in part account for nonproteasomal epitope generation as postulated for certain major histocompatibility complex class I alleles. In addition, TPPII may be able to substitute for some metabolic functions of the proteasome.

The specificity of proteasomes: impact on MHC class I processing and presentation of antigens.

We have studied polypeptide processing by purified proteasomes, with regard to proteolytic specificity and cytotoxic T-lymphocyte (CTL) epitope generation. Owing to defined preferences with respect to cleavage sites and fragment length, proteasomes degrade polypeptide substrates into cohorts of overlapping oligopeptides. Many of the proteolytic fragments exhibit structural features in common with major histocompatibility complex (MHC) class I ligands including fragment size and frequencies of amino acids at fragment boundaries. Proteasomes frequently generate definitive MHC class I ligands and/or slightly longer peptides, while substantially larger peptides are rare. Individual CTL epitopes are produced in widely varying amounts, often consistent with immunohierarchies among CTL epitopes. We further found that polypeptide processing is remarkably conserved among proteasomes of eukaryotic origin and that invertebrate proteasomes can efficiently produce known high-copy MHC class I ligands, suggesting evolutionary adaptation of the transporter associated with antigen processing and MHC class I to ancient constraints imposed by proteasomal protein degradation.

Potential immunocompetence of proteolytic fragments produced by proteasomes before evolution of the vertebrate immune system.

To generate peptides for presentation by major histocompatibility complex (MHC) class I molecules to T lymphocytes, the immune system of vertebrates has recruited the proteasomes, phylogenetically ancient multicatalytic high molecular weight endoproteases. We have previously shown that many of the proteolytic fragments generated by vertebrate proteasomes have structural features in common with peptides eluted from MHC class I molecules, suggesting that many MHC class I ligands are direct products of proteasomal proteolysis. Here, we report that the processing of polypeptides by proteasomes is conserved in evolution, not only among vertebrate species, but including invertebrate eukaryotes such as insects and yeast. Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes. Moreover, many major dual cleavage peptides produced by invertebrate proteasomes have the length and the NH2 and COOH termini preferred by MHC class I. Thus, the ability of proteasomes to generate potentially immunocompetent peptides evolved well before the vertebrate immune system. We demonstrate with polypeptide substrates that interferon gamma induction in vivo or addition of recombinant proteasome activator 28alpha in vitro alters proteasomal proteolysis in such a way that the generation of peptides with the structural features of MHC class I ligands is optimized. However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis. The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

Growth of wild-type and interferon-sensitive mengovirus in tk- L cells.

Cells lacking thymidine kinase (tk) have been reported to fail to respond to the action of interferon (IFN). While some laboratories have confirmed this observation, others have failed to do so. We studied the effect of IFN on four freshly isolated tk- lines of mouse L cells infected with mengovirus. In all cases, normal antiviral activity was induced. The antiproliferative activity of IFN was studied using the parental L cell line, a tk- derivative, and a tk- (tk+) subline into which the tk gene of herpes simplex virus was introduced. All three lines had a doubling time of about 20 h. In all cases, 2,000 U/ml of IFN increased this time to about 50 h. In contrast to the above results, an IFN-sensitive mutant of mengovirus (is-1) grew much better in protected tk- cells than in protected normal cells. This phenomenon appears to be dependent on the fact that tk- cells were routinely maintained in medium containing 5-bromodeoxyuridine (BUDR). In the absence of this drug, the virus behaved normally. The implications of this observation are discussed.

Attenuated live fowl cholera vaccine. I. Development of vaccine strain M3G of Pasteurella multocida.

A live cholera vaccine was developed from a virulent avian septicemia strain of Pasteurella multocida serotype 1. The virulent parental strain was mutagenized with N-methyl-N'-nitro-N-nitroso guanidine. Mutants were selected that had either smaller colonies at 37 C or temperature sensitivity for growth at 41 C. Four small-colony mutants and 2 temperature-sensitive mutants were studied. All the mutants were avirulent for turkeys. Sixteen days after turkeys were vaccinated with each mutant, both the vaccinates and unvaccinated controls were challenge-exposed to virulent P. multocida of the homologous serotype and the heterologous serotype 3. Two of the small-colony mutant strains protected against both homologous and heterologous challenge. Suggested for a live cholera vaccine is P. multocida M3G, a small-colony-forming mutant, innocuous for both mice and turkeys and stable against reversion.

Attenutated live fowl cholera vaccine. III. Laboratory and field vaccination trials in turkeys and chickens.

Administered via the drinking water, M-3-G, an attenuated strain of Pasteurella multocida of serotype 1, was found to immunize turkeys and chickens against fowl cholera. Immunity was tested by challenging birds intramuscularly, by palatine cleft swab, or orally after 3 vaccinations. No reactions to vaccination were noted in 390 turkeys in 12 laboratory trials, nor in 20,245 vaccinated in field trials. Chickens showed no vaccination reactions, and immunity was elicited by challenge in a laboratory trial and in face of natural outbreaks in the field, where 11,600 chickens were vaccinated. No vaccination reactions were noted, although most birds involved in the trials were carrying Mycoplasma spp. Immunity was found to last about 10 weeks after the last vaccination. The immunizing properties of M-3-G are compared with the CU strain.

Mallory bodies. Horseradish peroxidase: specific cytochemical and biochemical marker for alcoholic hyalin.

Horseradish peroxidase (HRP), a glycoprotein enzyme, bound specifically to Mallory bodies (MBs) in cryostat sections of autopsy liver and liver biopsies. In contrast, HRP did not bind to cryostat sections of normal liver. The specificity of HRP binding was also observed using light and electron microscopy in autopsy liver-derived subcell fractions prepared by the MB isolation procedure. In order to quantitate HRP binding, a solid phase colorimetric assay was developed. This assay involves immobilizing purified MBs or homogenized tissue fractions in glass tubes, incubating with HRP, and measuring the enzymatic activity of bound HRP. A linear relationship between MB concentration and HRP binding was observed. The assay was capable of detecting as little as 1 microgram of MB protein. The specificity of HRP binding was also investigated using the solid phase assay. The specific activity (HRP bound per milligram of protein) of purified MBs was 10 to 15 times that of a glass wool-filtered liver homogenate suggesting that the solid phase assay may be of use in monitoring the purification of MBs. HRP did not bind to normal liver homogenate even when large loads were assayed. The results of this study indicate that HRP binding, employed cytochemically, represents a rapid and facile procedure for ascertaining the presence of MBs in tissue. In some cases, those structures may not be easily visualized by conventional staining procedures. Furthermore, quantitation of MBs in tissue may be possible by using a solid phase enzyme-linked assay.

Mallory bodies: isolation of hepatocellular hyalin and electrophoretic resolution of polypeptide components.

Mallory bodies (MBs) were obtained in purified form from human liver obtained at autopsy using a new procedure consisting of sedimentation through a Ficoll viscosity barrier. Preparations from six livers ranged in purity from 95 to 99 per cent. MB preparations were autofluorescent. MBs were strongly agglutinated by Concanavalin A. The presence of carbohydrate was also indicated by the fact that MBs bound fluorescently labeled Concanavalin A; no binding was observed in the presence of appropriate inhibitor monosaccharides. Direct analysis indicated that MBs contained variable amounts of neutral hexose (0.65 to 2.4 mumoles of glucose-equivalents per milligram of protein) but no sialic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that purified MBs contain five major polypeptides possessing apparent molecular weights of 56,000, 48,500 to 45,000 (triplet), and 32,500. Periodic acid-Schiff-positive components were not detected. Scanning electron microscopy of isolated MBs revealed the presence of a rough, fibrous surface, whereas conventional transmission electron microscopy indicated the filamentous nature of MBs.