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BACKGROUND: Reduction in pulmonary exacerbations is an important efficacy endpoint for CF clinical studies. Powering exacerbation endpoints requires estimation of the future exacerbation incidence in CF study populations, but rates differ across the population. METHODS: We have estimated exacerbation rates for Epidemiologic Study of CF subpopulations stratified by age, FEV(1)% predicted, sex, weight-for-age percentile, respiratory signs and symptoms, and history of exacerbation and bacterial culture. Sample sizes required to attain 80% power to detect exacerbation reductions of 20% to 80% in 1:1 randomized studies of 3 to 12 month duration were determined. Exacerbation treatments with "any" antibiotic (new oral quinolone, new inhaled antibiotic, or intravenous (IV) antibiotic) and with IV antibiotics were studied. RESULTS: At all ages, decreased FEV(1), female sex, exacerbation history, and Pseudomonas aeruginosa culture history were associated with increased treatment for exacerbation. CONCLUSIONS: These data should assist investigators in the design of future CF exacerbation studies.

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BACKGROUND: Rate of lung function decline (RLFD) (as FEV(1) percent predicted/yr) is a robust measure of CF therapeutic efficacy rarely used as a study endpoint, in part due to uncertainty of sample size requirements. METHODS: Sample size requirements for 1:1 randomizations to detect RLFD treatment effects from 20% to 80% were assessed in Epidemiologic Study of CF (ESCF) patients. Effects of measuring FEV(1) 1-4 times per year in studies of 1- to 4-year durations were assessed in 399 patients age ≥ 6 years with FEV(1) ≥ 70%. Impacts of inclusion/exclusion based on risk factors in 2369 ESCF patients were assessed over 1.5 years using semi-annual FEV(1) measures. RESULTS: Increasing study duration and exclusion of lower risk patients (e.g., no P. aeruginosa infection) both substantially reduced requirements. CONCLUSIONS: CF RLFD studies of 1.5 years in duration appear feasible provided that investigators account for the beneficial effects of subject inclusion/exclusion based on risk factors in power estimates.

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Slices of porcine M. longissimus dorsi were packed in overwrap packs and subjected to irradiation (0 and 5 kGy) and then stored for 7 days at 4°C. Reflectance spectra were measured on the outside surface and a freshly cut surface at 7 days post irradiation. The reflectance spectra were transformed to reflex attenuance, k/s and first and second difference spectra and subjected to discriminant analysis. Using discriminant analysis it was possible to establish a calibration equation to discriminate between the spectra of irradiated and unirradiated pork for both the outside and the inside surface. When the calibration model was used to predict the classification of new samples a 100% correct grouping was obtained for the freshly cut surface, however, for the outside surface the classification ranged from 87 to 100% correct depending on the mathematical transformation of the reflectance spectra. This shows the potential of colour measurements as a possible rapid initial screening test for the identification of irradiated pork. Evaluation of the first difference spectra to identify peak positions showed significant differences in peak positions between irradiated and unirradiated pork. The position of the peaks in the irradiated sample is discussed as lending support to the hypothesis of the carboxyhaem form as the irradiated pigment.

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The effect of irradiation (0 and 5 kGy) of beef, pork and lamb portions in retail overwrap packs and subsequent storage at 4°C was studied in relation to colour changes. The colour of the exterior surface of beef and pork was measured on the same samples on each day of storage for up to 7 days post irradiation. On day 7 the colour of a freshly cut surface was measured. The colour of both the exterior and a freshly cut surface of lamb, in similar retail overwrap packs was measured at 2, 5 and 7 days, post irradiation, different samples being used on each day of measurement. L* values of irradiated beef increased significantly with storage and a* values for unirradiated samples decreased significantly with storage. For lamb there was a general increase in L* and h(o) values and a decrease in a*, b* and C* values with storage. Analyses of the day 7 data showed statistically significant effects for species on all CIELAB parameters. Irradiation resulted in significantly higher hue angle (h(o)) values and the a*, b* and C* values were significantly higher on the exterior than freshly cut surface. There were a number of statistically significant 2 factor and 3 factor interactions. The role of formation of a carboxyhaem pigment in the colour of irradiated meat is discussed. The problem of interpretation of pigment changes from CIELAB values is highlighted.

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The effect of irradiation (0 and 5 kGy) of chicken, goose and turkey breast and leg muscles and subsequent storage at 4°C was studied in relation to colour changes. The colour of the outside surface was measured on the breast on each day of storage for up to 7 days post irradiation and for breast and leg and day 7. The colour of a freshly cut interior surface of both breast and leg was measured after 7 days storage. L* values of control and irradiated chicken, goose and turkey breast muscles changed little during storage post irradiation. The a* values for unirradiated goose breast were significantly higher than irradiated goose breast but declined to values similar to irradiated goose breast after 7 days of storage. The b* values for irradiated turkey breast were significantly higher than unirradiated turkey breast at all times post irradiation treatment. Analysis of variance was performed on the day 7 CIELAB values of breast muscle for the effects of species, surface and irradiation and their interactions. After 7 days storage a* values of poultry breast were higher on the freshly cut surface due to irradiation in all species, with decreases in hue angle due to irradiation. The a* values of leg of all species at 7 day post irradiation was significantly higher in the irradiated treatment than the controls. The results for the turkey leg indicate that this effect may be mainly due to higher a* values of the freshly cut surface. The possible role of carboxy form of the haem pigments as the irradiated pigment form is discussed.

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The three principal myoglobin states of haem pigment, oxymyoglobin, metmyoglobin reduced myoglobin, and the ferrous nitrous oxide form, nitrosomyoglobin, were prepared as pure pigments in solution. The absorbance spectra of these solutions were determined and extinction coefficients calculated. The position of the absorption peaks showed some small differences compared to published data, in particular the use of absorbance readings at 525 nm as an isobestic point for all three could be questioned. The use of mathematical transforms to first or second difference functions showed merit for the identification of mixtures of some of the myoglobin forms. The second difference function separated the broad absorbance band in the Soret region into two separate troughs, thereby resolving a mixture of oxymyoglobin and reduced myoglobin. It is suggested that based on absorption spectra, similar mathematical transforms could be applied to the interpretation of reflectance spectra of meat and meat products. Further evaluation of such mathematical transformations is required on a range of meat reflectance spectra.

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The family of type 2 cystatin proteins are a class of cysteine proteinase inhibitors found in a variety of human fluids and secretions, where they appear to provide protective functions. To establish the size of the human gene family encoding these proteins, we isolated cosmid and lambda genomic clones. Restriction mapping, partial sequence analysis, and hybridization studies identified a total of seven distinct genes, six of which correspond to known genes and proteins. Sequence analysis showed that the seventh gene, CSTP2, is an apparent pseudogene carrying a nonsense mutation in exon 1 distinct from that in CSTP1. Southern blots of genomic DNA probed with gene-specific probes accounted for all but one or two sets of fragments containing exon 1, and one or two sets of fragments containing exon 3, indicating that the human type 2 cystatin gene family consists of eight or nine members. Southern blot analysis of large restriction fragments using these gene-specific probes indicates that all seven of the cloned type 2 cystatin genes are clustered at a single locus on human chromosome 20. This locus is no larger than about 910 kb, and possibly as small as 365 kb. We designate this as the CST locus and suggest a numbering system for the cystatin genes.

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Humans carry one gene encoding cystatin C and six to eight genes with homology to an S-like cystatin hybridization probe. However, the precise composition and organization of the cystatin gene family remains to be established. Further, the pattern of tissue-specific expression has not been fully defined. We have previously shown that the type 2 cystatin genes are clustered together in a ca. 270 kb region (the CST locus). To determine the structure of this region, we have sought to clone the entire CST locus. Our approach has been to isolate cosmid and lambda genomic clones carrying cystatin genes and then to use "walk" probes derived from the end regions of these clones to identify other clones, which extend them. To date, we have obtained over 320 kb of distinct sequences. Based on restriction maps, sequencing, and hybridization analyses, we have identified eight apparently nonallelic copies of cystatin genes. These include one gene for cystatin C, four closely related genes encoding S-like cystatins, and three genes encoding relatively divergent sequences. Complete assembly of these clones into an unambiguous contiguous sequence is hampered by the presence of flanking locus-specific repetitive-like sequences. RNase protection assays used to characterize the tissue-specific patterns of expression showed that cystatin C is expressed at modest, comparable levels in all tissues examined, whereas expression of the CST 1 gene, encoding cystatin SA-I, was found to be restricted to a small subset of tissues, with the highest level in the submandibular gland.(ABSTRACT TRUNCATED AT 250 WORDS)

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Certain genomic sequences cannot be recovered efficiently in cosmid or lambda bacteriophage clones, presenting a barrier to efforts to construct a contiguous cloned library of a genome. We have encountered such sequences during our efforts to isolate cosmid and bacteriophage lambda clones carrying members of the human type 2 cystatin gene family. Several cosmid clones constructed in the pWE 15 vector did not survive purification, and using standard techniques, we were unable to obtain significant amounts of cosmid DNA from those clones we could purify. Similarly, several lambda bacteriophage clones constructed in the lambda DASH II vector could not be purified, and those lambda clones we were able to isolate gave low titers in liquid lysates. In this paper, we describe generally applicable methods for preparing high yields of recombinant DNA from such recalcitrant cosmid and lambda clones constructed in these vectors.

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The effect of lipopolysaccharide preparations from Salmonella enteritidis, Bacteroides gingivalis, and Actinobacillus actinomycetemcomitans on human gingival fibroblasts was studied. Lipopolysaccharide from all sources inhibited fibroblast proliferation in the concentration range of 0.5 to 50 micrograms/ml, with the lipopolysaccharide from A. actinomycetemcomitans having the strongest inhibitory effect. Assessment of the effect of lipopolysaccharide on gingival fibroblast metabolism indicated both total protein and proteoglycan synthesis to be inhibited with increasing concentrations of lipopolysaccharide. As for the antiproliferative effect, lipopolysaccharide from A. actinomycetemcomitans had the greatest inhibitory effect on cell synthetic activity. This inhibitory effect was determined by pulse-chase experiments to be a true depression in synthesis. Furthermore, the effect was independent of lipopolysaccharide-induced changes in cell proliferation and prostaglandin synthesis. This study confirmed the toxic effect of lipopolysaccharide on fibroblasts and, in particular, indicated that various lipopolysaccharide preparations vary in their potency to influence cell proliferation and extracellular matrix synthesis.

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A monoclonal antibody, BBG-25, raised in BALB/c mice demonstrated specificity for Bacteroides gingivalis lipopolysaccharide. Immunoblotting indicated that this monoclonal antibody does not cross-react with lipopolysaccharide prepared from enterobacterial organisms or from other Bacteroides species.

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BALB/c mice were immunized with an invasive (A7A1-28) or noninvasive (381) Bacteroides gingivalis strain, Bacteroides intermedius, or Ringer solution. All immunized mice were subsequently challenged with the invasive B. gingivalis strain and examined for septicemia or secondary spread of the infection or both. Mice immunized with the invasive B. gingivalis strain localized the infection to the challenge site. Mice immunized with the noninvasive B. gingivalis strain, B. intermedius, or Ringer solution developed spreading infections. These data suggest that immunization with an invasive B. gingivalis strain can alter the course of subsequent infections.

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The ultrastructures and surface protein profiles of aerobically cultured Actinobacillus actinomycetemcomitans (Haemophilus actinomycetemcomitans) differed from those of cells cultured anaerobically. Similar ultrastructural differences were also observed when aerobic and anaerobic cultures of a strain of Escherichia coli were compared. These results suggest that oxygen-related variations in the bacterial cell surface may play a role in the adaptation of oral bacteria to different host environments.

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Lipoteichoic acid (LTA), extracted from Streptococcus mutans 10449 by hot aqueous phenol, was partially purified by Sepharose 6B column chromatography in 0.01 M sodium acetate, pH 6.0, containing 0.25 M sodium chloride and 0.001 M EDTA. Nucleic acid and polysaccharide were precipitated from the LTA-containing column peak by the addition of 2 volumes of chloroform-methanol (1:5). The resulting single-phase chloroform-methanol-water (1:5:3) supernatant contained LTA and small amounts of several contaminating substances as indicated by reverse-phase high-pressure liquid chromatography and chemical analyses. LTA was purified further by DEAE-cellulose chromatography, using a concentration gradient of sodium chloride in chloroform-methanol-water (1:5:3). Two column peaks of LTA were found to contain phosphate, glycerol, glucose, and fatty acids at molar ratios of 1:1:0.11:0.10 and 1:1:0.09:0.04, respectively. The LTA polymers contained 18 and 22 repeating units of unsubstituted glycerophosphate and two glucose residues. The LTA in one column peak had two fatty acids per molecule, whereas that in the second peak contained only one. The yield of LTA was 1.68 mg per g of cell dry weight or 65 mg per g of phenol-water-extracted material. The specific activity of the LTA preparation was increased 128-fold by the purification scheme as determined by a erythrocyte-binding assay. Reverse-phase high-pressure liquid chromatography may be used for rapid separation of LTA molecules containing different numbers of acyl groups.

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Two separate species of lipopolysaccharide (LPS) from Bacteroides gingivalis 381 have been isolated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated not only the heterogeneity of each species, but also that they represented high- and low-molecular-weight LPS entities. Although both contained the same carbohydrate and fatty acid components, the proportions of these differed between the LPS species. The direct effects of these two species in modulation of bone resorption and bone collagen and noncollagen protein synthesis have been examined. In a bone resorption assay, these two species stimulated 45Ca release from prelabeled fetal rat bones in a concentration range of 0.5 to 3.0 micrograms/ml. The two LPS species also elicited a 30 to 40% reduction in collagen protein formation at 10 micrograms/ml. Responses of the same order of magnitude were observed with LPS from Salmonella minnesota at 10 micrograms/ml. The higher-molecular-weight LPS species also significantly inhibited noncollagen protein formation. This is the first report that LPS from B. gingivalis 381, a suspected periodontal pathogen, inhibits bone collagen formation and, in conjunction with the bone resorption potency, further implicates LPS in alveolar bone loss associated with periodontal disease.

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In antral mucosa of the mouse stomach, the volume of mucus in mucous cells was measured morphometrically to determine whether this value changes during cell migration from the base of the pit to the surface. Both the volume density of mucous granules (the fraction of cell volume occupied by the granules) and the volume of intracellular mucus were reduced to about half in surface cells compared with those of upper pit cells. This indicates that mucus secretion is substantial during the later part of the lifespan of these cells, and is not due simply to the shedding of senescent cells.

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Measurement of gastric mucus production in vivo poses several problems. We therefore used organ culture of rat antrum to measure mucosal synthesis and secretion of glycoprotein. Tissue maintained good viability for 20 hr. Glycoprotein synthesis rate, assessed by incorporation of [3H]fucose and [3H]glucosamine, was constant during 20 hr. [3H]Fucose incorporation increased in proportion to increasing [fucose] in medium until the latter reached 10(-3) M, then the slope decreased. Cycloheximide abolished [3H]fucose incorporation into glycoprotein. Two methods for measuring secretion gave parallel results: prelabeled glycoprotein was secreted linearly, with 60% of pulse-labeled tissue glycoprotein appearing in medium in 20 hr. The data suggest that (1) antral mucous cells contain a high-concentration pool of endogenous fucose, but possibly only a small-capacity pool of preformed apoglycoprotein, (2) synthesis and secretion of glycoprotein can be successfully examined with organ culture, and (3) secretion in vitro occurs at a steady, slow rate.

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