Determinants of between-hospital variations in outcomes for patients admitted with COPD exacerbations: findings from a nationwide clinical audit (AUDIPOC) in Spain.

BACKGROUND: Previous studies have demonstrated significant variability in the processes of care and outcomes of chronic obstructive pulmonary disease (COPD) exacerbations. The AUDIPOC is a Spanish nationwide clinical audit that identified large between-hospital variations in care and clinical outcomes. Here, we test the hypothesis that these variations can be attributed to either patient characteristics, hospital characteristics and/or the so-called hospital-clustering effect, which indicates that patients with similar characteristics may experience different processes of care and outcomes depending on the hospital to which they are admitted. METHODS: A clinical audit of 5178 COPD patients consecutively admitted to 129 Spanish public hospitals was performed, with a 90-day follow-up. Multilevel regression analysis was conducted to model the probability of patients experiencing adverse outcomes. For each outcome, an empty model (with no independent variables) was fitted to assess the clustering effect, followed by a model adjusted for the patient- and hospital-level covariables. The hospital-clustering effect was estimated using the intracluster correlation coefficient (ICC); the cluster heterogeneity was estimated with the median odds ratio (MOR), and the coefficients of predictors were estimated with the odds ratio (OR). RESULTS: In the empty models, the ICC (MOR) for inpatient mortality and the follow-up mortality and readmission were 0.10 (1.80), 0.08 (1.65) and 0.01 (1.24), respectively. In the adjusted models, the variables that most represented the patients' clinical conditions and interventions were identified as outcome predictors and further reduced the hospital variations. By contrast, the resource factors were primarily unrelated with outcomes. CONCLUSIONS: This study demonstrates a noteworthy reduction in the observed crude between-hospital variation in outcomes after accounting for the hospital-cluster effect and the variables representing patient's clinical conditions. This emphasises the predictor importance of the patients' clinical conditions and interventions, and understates the impacts of hospital resources and organisational factors.

Prediction of dairy housing construction costs.

Dairy farms in Galicia and elsewhere in Europe are going through a transition phase to adapt to modern dairy technology, improve labor efficiency, and increase in size and scale. Expanding a dairy herd and building housing for more cows can be very expensive. A poor decision during expansion can result in serious financial difficulties even to the point of making the farm economically unviable. Dairy managers must carefully evaluate existing alternatives and must select an optimal strategy. To aid this decision, a computer spreadsheet application has been developed that predicts the cost per cow and cost per unit of area of alternative designs as functions of the number of cows to be housed. The spreadsheet is, in principle, applicable to a wide variety of designs and to housing for livestock other than dairy cattle. However, the current database allows comparison among six of the dairy housing designs that have been used most widely in Galicia in recent years. From projected financial results of the developed model, it was concluded that differing designs were preferred for different farm circumstances. Preferred designs for farms with 60 to 200 cows were either four rows of facing free stalls or four rows of tail-to-tail free stalls, which have virtually the same costs. Whereas for farms with fewer than 60 cows, the preferred design was two rows of tail-to-tail free stalls, designs with three rows of free stalls were generally more costly per cow. Results of design calculations must be integrated with other farm management considerations in choosing a particular design.

Activation of a cryptic 5' splice site by U1 snRNA.

In the course of analyzing 5' splice site mutations in the second intron of Schizosaccharomyces pombe cdc2, we identified a cryptic 5' junction containing a nonconsensus nucleotide at position +2. An even more unusual feature of this cryptic 5' junction was its pattern of activation. By analyzing the profile of splicing products for an extensive series of cdc2 mutants in the presence and absence of compensatory U1 alleles, we have obtained evidence that the natural 5' splice site participates in activation of the cryptic 5' splice site, and that it does so via base pairing to U1 snRNA. Furthermore, the results of follow-up experiments strongly suggest that base pairing between U1 snRNA and the cryptic 5' junction itself plays a dominant role in its activation. Most remarkably, a mutant U1 can activate the cryptic 5' splice site even in the presence of a wild-type sequence at the natural 5' junction, providing unambiguous evidence that this snRNA redirects splicing via base pairing. Although previous work has demonstrated that U5 and U6 snRNAs can activate cryptic 5' splice sites through base pairing interactions, this is the first example in which U1 snRNA has been implicated in the final selection of a cryptic 5' junction.

Syllables and morphemes: contrasting frequency effects in Spanish.

Three types of sublexical units were studied in Spanish visual word recognition: the syllable, the basic orthographic syllabic structure (BOSS), and the root morpheme. In Experiment 1, using a lexical-decision task, a typical inhibitory effect of the first-syllable frequency was found (while keeping constant the BOSS frequency) as well as the word-frequency effect. Experiment 2 examined the role of both the BOSS frequency and the word frequency, also in a lexical-decision task. Syllable frequency was controlled. Both the BOSS frequency and the word frequency showed facilitatory effects. However, in Experiments 3A and 3B, a facilitatory effect of the root frequency (when controlling for BOSS frequency) and a null effect of BOSS frequency (when controlling for root frequency) were found, suggesting that the BOSS effect is in fact reflecting a morpheme effect. A review of the current models shows that it is difficult to integrate syllables and morphemes in a unique model.

Evidence for splice site pairing via intron definition in Schizosaccharomyces pombe.

Schizosaccharomyces pombe pre-mRNAs are generally multi-intronic and share certain features with pre-mRNAs from Drosophila melanogaster, in which initial splice site pairing can occur via either exon or intron definition. Here, we present three lines of evidence suggesting that, despite these similarities, fission yeast splicing is most likely restricted to intron definition. First, mutating either or both splice sites flanking an internal exon in the S. pombe cdc2 gene produced almost exclusively intron retention, in contrast to the exon skipping observed in vertebrates. Second, we were unable to induce skipping of the internal microexon in fission yeast cgs2, whereas the default splicing pathway excludes extremely small exons in mammals. Because nearly quantitative removal of the downstream intron in cgs2 could be achieved by expanding the microexon, we propose that its retention is due to steric occlusion. Third, several cryptic 5' junctions in the second intron of fission yeast cdc2 are located within the intron, in contrast to their generally exonic locations in metazoa. The effects of expanding and contracting this intron are as predicted by intron definition; in fact, even highly deviant 5' junctions can compete effectively with the standard 5' splice site if they are closer to the 3' splicing signals. Taken together, our data suggest that pairing of splice sites in S. pombe most likely occurs exclusively across introns in a manner that favors excision of the smallest segment possible.

The expression of drug resistance gene products during the progression of human prostate cancer.

Prostate cancer progresses from a localized disease to a widely disseminated malignancy. Each step along this progression pathway involves multiple genetic alterations that impart a survival advantage to the tumor cell over its normal counterparts and may confer resistance to therapy. Because metastatic prostate cancer is one of the most therapy-resistant human neoplasms, we studied the expression of certain molecular determinants of drug resistance in the context of tumor progression. Paraffin-embedded formalin-fixed resected prostates were chosen based on Gleason grade and surgical stage. Immunohistochemistry was used to detect the expression of multidrug resistance protein (MRP), topoisomerase II alpha, p53, glutathione S-transferase pi, Bcl-2, and P-glycoprotein in these specimens. We found that all of the proteins were expressed in resected prostate except for P-glycoprotein. The expression of MRP, topoisomerase II alpha, p53, and Bcl-2 increased with the Gleason grade. In addition, the expression of MRP, topoisomerase II alpha, and p53 increased with the surgical stage. In contrast, the glutathione S-transferase pi and Bcl-2 expression decreased with the increasing surgical stage. Stage was the strongest indicator of protein expression. These results suggest that drug resistance gene products are expressed in prostate cancer at the time of surgical resection. Thus, although the emergence of the "pan-resistance" phenotype in prostate cancer may partly be a function of the selection pressure exerted by therapeutic interventions, certain determinants of chemoresistance may be caused by genetic changes accompanying tumorigenesis.

Prostaglandin H synthase expression is variable in human colorectal adenocarcinoma cell lines.

The expression of prostaglandin H synthases can be induced by many stimuli and is likely to be important in control of the cell cycle. The analysis of prostaglandin H synthase-1 and -2 expression in colon adenocarcinoma cell lines is a useful model system for studying the function of the prostaglandin H synthases, especially with regard to proliferation and adhesion. Prostaglandin H synthase-1 protein is not found in any of eight human colon adenocarcinoma cell lines. Expression of prostaglandin H synthase-2 is variable for the eight cell lines: three constitutively expressed active protein, four did not express this gene at all, and one had mRNA but no active protein. Thus, five colorectal adenocarcinoma cell lines exhibit "null" expression of prostaglandin synthase-2. The three cell lines with constitutive expression of prostaglandin H synthase-2 produce PGE2. Prostaglandin E2 production could be inhibited by aspirin and NS398 without inhibiting proliferation, while direct addition of prostaglandin E2 inhibits proliferation. Adhesion to collagen IV and fibronectin was stronger in those cell lines that expressed prostaglandin H synthase-2. The constitutive expression of prostaglandin H synthase-2 is associated with increased adhesion to extracellular matrix components and a potential inhibition of proliferation through the production of prostaglandin E2. The absence of PGH synthase-2 expression in some cell lines may result from the original tumor's need to inactivate these associated functions. Our evidence suggests that PGH synthase-2 is a possible candidate for a tumor suppressor gene at 1q23-qter.

Mutational analysis of U1 function in Schizosaccharomyces pombe: pre-mRNAs differ in the extent and nature of their requirements for this snRNA in vivo.

The U1 snRNP is known to play a critical role in spliceosome assembly, at least in part through base pairing of its RNA moiety to the substrate, but many details remain to be elucidated. To further dissect U1 snRNA function, we have analyzed 14 single point mutations in the six nucleotides complementary to the 5' splice site for their effects on growth and splicing in the fission yeast Schizosaccharomyces pombe. Three of the four alleles previously found to support growth of Saccharomyces cerevisiae are lethal in S. pombe, implying a more critical role for the 5' end of U1 in fission yeast. Furthermore, a comparison of phenotypes for individual nucleotide substitutions suggests that the two yeasts use different strategies to modulate the extent of pairing between U1 and the 5' splice site. The importance of U1 function in S. pombe is further underscored by the lethality of several single point mutants not examined previously in S. cerevisiae. In total, only three alleles complement the U1 gene disruption, and these strains are temperature-sensitive for growth. Each viable mutant was tested for impaired splicing of three different S. pombe introns. Among these, only the second intron of the cdc2 gene (cdc2-I2) showed dramatic accumulation of linear precursor. Notably, cdc2-I2 is spliced inefficiently even in cells containing wild-type U1, at least in part due to the presence of a stable hairpin encompassing its 5' splice site. Although point mutations at the 5' end of U1 have no discernible effect on splicing of pre-U6, significant accumulation of unspliced RNA is observed in a metabolic depletion experiment. Taken together, these observations indicate that the repertoire of U1 activities is used to varying extents for splicing of different pre-mRNAs in fission yeast.

Proliferation of germinal center B lymphocytes in vitro by direct membrane contact with follicular dendritic cells.

In secondary lymphoid organs, follicular dendritic cells (FDC) are located within B cell follicles and germinal centers. Through their cytoplasmic extensions they come into contact with a large number of neighboring lymphocytes. Using an enzyme cocktail to digest human tonsils followed by ultracentrifugation on bovine serum albumin gradients, single cell suspensions were obtained. Immunocytochemistry revealed that 7% of the cells were FDC, 5% T cells, and 5% macrophages. The remaining population were B cells with greater than 95% being of the germinal center phenotype (i.e. CD19-positive, CD39/sIgD negative). After 24 h of culture up to 44% of the lymphocytes were found in clusters centered around FDC. At the start of the culture as well as 24 and 72 h later, between 31 and 55% of the B cells within FDC associated clusters were in late G1 to M phase of the cell cycle. In contrast, less than 10% of the B cells not in contact with FDC (i.e. outside the clusters) were in an activated state. Autoradiography revealed that after three days of incubation the rate of proliferation was 26.2 times higher for the lymphocytes involved in cluster formation as compared to those cells not associated with FDC. Furthermore, the number of viable B cells after a 72 h mitogen-free culture period was determined. By adding FDC to these preparations, 31.9% of the lymphocytes were rescued from dying. These data show that FDC provide a microenvironment which can maintain the viability, activation and proliferation of germinal center B cells in vitro.

Ultrastructural cytochemistry and radioautography of complex carbohydrates in heterophil granulocytes from rabbit bone marrow.

The subcellular route of incorporation of complex carbohydrates into rabbit heterophil primary granules and their subsequent intragranular distribution during granule maturation were studied with ultrastructural, cytochemical, and radioautographic methods. High iron diamine (HID) staining of sulfated glycoconjugates in primary granules was partially diminished after treatment with chondroitinase ABC or after removal of N-sulfate groups with nitrous acid, but was not altered by exposure to hyaluronidase, trypsin, or HCl. Subsequent thiocarbohydrazide-silver proteinate (TCH-SP) straining of thin sections increased the density of the HID reaction product. Golgi-derived spherules and very immature morular granules stained weakly with HID-TCH-SP and labeled intensely after a 10 min incubation with 35SO4. After a 60 min 35SO4 pulse and a 60 min chase, an increase in radiolabeling was observed in granules with HID stained, fused morular material, and some labeling was present in more mature rim stained granules. Fully mature granules lacked HID or HID-TCH-SP staining, but contained most of the 35SO4 labels after a 60 min pulse and 18 hr chase in vitro. Periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of unosmicated thin sections localized vicinal glycol-containing complex carbohydrates in Golgi-associated small vesicles. These vesicles lacked HID-TCH-SP staining and apparently contained neutral glycoprotein. They frequently bordered, in a rosette arrangement, the immature morular granules, but not the more mature primary granules. The PA-TCH-SP method localized complex carbohydrates in the rim of granules precursors and enclosed a spherule or morula, but failed to stain the sulfate-containing material in the morulas or spherules. PA-TCH-SP reactivity was diffusely distributed in moderately mature granules and was decreased in fully mature granules. These results indicate that heterophil primary granule contain several complex carbohydrates including O-sulfated and N-sulfated glycosaminoglycans, as well as vicinal glycol-containing glycoproteins. These complex carbohydrates are transported to immature primary granules by different Golgi-derived organelles. The complex carbohydrates are subsequently distributed differently within primary granules and become masked to staining as the granule matures.

Ferrocyanide enhancement of concanavalin A-ferritin and cationized ferritin staining blood cell surface glycoconjugates.

Ferrocyanide was used to enhance cationized ferritin and concanavalin A-ferritin (Con A-ferritin) staining of surface glycoconjugates of peripheral blood and bone marrow cells from rabbits and humans. The glutaraldehyde-fixed cells were stained with Con A-ferritin or cationized ferritin and then exposed to a ferrocyanide solution. The resulting cuboidal and irregular stain deposits averaged 50 nm in diameter when viewed with the transmission (TEM) and scanning electron microscope (SEM). Rabbit blood cells demonstrated more Con A binding sites than human blood cells and the decrease in binding sites observed with maturation of human granulocytic and erythrocytic cells was not evident in rabbit cells. Differences in binding of cationized ferritin to rabbit and human cell surfaces were less prominent than that observed for Con A. These results extend previous studies of blood cell surface glycoconjugates and demonstrate that ferrocyanide enhancement significantly facilitates SEM evaluation of Con A-ferritin and cationized ferritin bound to cell surfaces.

Ultrastructural localization of nonheme celluar iron with ferrocyanide.

The Prussian blue reaction was evaluated at the ultrastructural level as a cytochemical method to identify ferric and ferrous iron in rat bone marrow and splenic macrophages. Satisfactory tissue preservation and staining were achieved after fixation for 1 hr in 3% glutaraldehyde and exposure for 30 min to Perls's ferrocyanide solution before routine osmication and embedding. The acid ferrocyanide solution formed cuboidal and irregular electron-opaque deposits which localized ferric iron in the macrophage siderosomes and hyaloplasm. When thin sections were directly stained with the acid ferrocyanide, the stain deposits were much less distinct. The size and number of cytes exhibited sparse evenly distributed stain deposits. Several cells displayed abundant precipitates on the inner surface of the plasmalemma. Prussian blue precipitates were occasionally seen in mitochondria and nuclear euchromatin. Although osmium tetroxide post-fixation improved tissue preservation, it did not enhance the density of the ferri-ferrocyanide precipitate. The ferrocyanide solution yielded cuboidal deposits also in clots impregnated with ferritin, and electron diffraction analysis confirmed the symmetrical crystal structure of these stain precipitates. Smaller irregular precipitates were formed in clots impregnated with FeCl3, or Fe2 (SO4)3 solutions, despite the equally interpreted as indicating that the iron hydroxide core or protein structure of ferritin and hemosiderin contributed to the formation of the ultrastructurally evident cuboidal precipitates, but were not necessary for the formation of a colored reaction product. The acid ferrocyanide solution failed to stain clots formed in FeCI2, CuCI2 or CuCI solutions. Staining with a ferricyanide solution identified only sparse foci of ferrous iron in some siderosomes. This study demonstrates that the Prussian blue reaction can be used ultrastructurally to localize iron cations bound to some nonheme iron binding proteins.

Vitamin A deficiency and new bone growth: histologic changes.

Using a standardized guinea pig model system, the histologic appearance of newly formed bone from vitamin A deficient, vitamin A adequate and retinoic acid treated animals was compared. Microscopic examination of the histologic sections of bone collected from vitamin A deficient guinea pigs indicated the presence of highly cellular and loosely woven bone spicules. This model system is an osteoblastic model, since osteoclasts are essentially absent in tissue formed in 14-day implants. Thus, new bone formation in the vitamin A deficient guinea pig reveals morphologic changes which are principally mediated through the osteoblastic process.