In situ characterization of human cytomegalovirus infection of bronchiolar cells in human transplanted lung

Distal airway cell infection by human cytomegalovirus (HCMV) in transplanted lung has been occasionally reported but not systematically investigated. The present study aimed at testing the prevalence of HCMV bronchiolar infection in human transplanted lung. We identified and immunophenotyped, with double labeling, infected lung cells in 31 transbronchial biopsies with HCMV infection, containing distal airways (7 HCMV pneumonias, 7 HCMV infection without inflammation, and 17 morphologically occult, non-cytopathic HCMV infection). HCMV-infected cells in pneumonias, localizations, and occult infections were alveolar epithelia (32.8%, 42.8%, and 53.5%, respectively), endothelia (22.9%, 24.7%, and 26.4%, respectively), macrophages (0.006%, none, and none, respectively), airway epithelia (0.01%, 8.9%, and none, respectively), and bronchiolar smooth muscle cells (0.011%, 14.6%, and 16.1%, respectively). Ciliated and bronchiolar smooth muscle cells in transplanted lung only occasionally harbored viral infection and never showed viral cytopathy. On the basis of our morphological observations, HCMV infection of bronchiolar wall cells is rare, while alveolar epithelia and capillary endothelial cells are the major targets of lung infection.     

Neural precursor proliferation and newborn cell survival in the adult rat dentate gyrus are affected by vitamin E deficiency

The adult hippocampal neurogenesis is affected by vitamin E deficiency. In the present investigation we examined if neural precursor proliferation, newborn cell survival or both are altered by vitamin E deficiency. 5-Bromo-2'-deoxyuridine (BrdU) was employed as a marker of proliferating cells. BrdU-labelled cells were revealed 1 and 30 days after BrdU administration in order to evaluate proliferation and newborn cell survival, respectively. Cell proliferation decreased in controls from juvenile to adult age, and the decrease was lesser in vitamin E deficiency. Thus we found a higher number of proliferating cells in vitamin E-deficient rats than in age-matched controls at 5 months of age. Comparing the number of BrdU-positive cells between 1 and 30 days after the last BrdU injection revealed a remarkable decrease in all groups; this is the greatest in vitamin E-deficient rats and the lowest in control rats. Consistently cell death in the dentate gyrus, assessed by TUNEL technique, was found to decrease from 1 to 5 months of age, but at 5 months it was significantly higher in vitamin E-deficient rats than in age-matched controls. These data show that vitamin E deficiency enhances neural precursor proliferation and cell death during adult neurogenesis.     

Analysis of macrolide-lincosamide-streptogramin B (MLS(B)) resistance determinant in strains of Clostridium difficile

The macrolide-lincosamide-streptogramin B (MLSB) resistance determinants have been detected among Clostridia in both C. perfringens and C. difficile strains. Previous studies have shown that MLSB-resistant C. difficile strains can be differentiated by specific hybridizing bands using an erm(B) probe. A recent study has demonstrated that C. difficile 630, a strain highly resistant to clindamycin and erythromycin (MIC > or = 256 ml/L), showing a hybridizing band at 9.7 kb, contains two copies of an erm(B) gene. It was also hypothesized that C. difficile 630 erm(B) determinant has arisen from a progenitor, represented by the C. perfringens CP592 determinant, which contains only one copy of an erm(B) gene that differs from C. difficile 630 erm(B) for seven nucleotide substitutions. To investigate the possibility that C. difficile strains with hybridizing fragments of different molecular size have an erm(B) determinant not identical to the one described in C. difficile 630, we performed a genetic analysis on the erm(B) determinant in 18 C. difficile strains, isolated from different sources. The results showed a heterogeneity in erm(B) determinant: C. difficile strains with hybridizing bands at 7.3 or 3.7 kb contained only one erm(B) copy, whereas strains with a band at 9.7 kb had two copies. The majority of the toxigenic strains examined was characterized by only one erm(B) copy with a sequence identical to the one found in C. difficile 630 and a lower resistance level for erythromycin (MICs ranging from 16 to 24 ml/L). Differently, some strains had an erm(B) gene identical to the one found in C. perfringens CP592. PCR ribotyping and clustering analysis indicate that the examined resistant strains, except one, belong to the same genetic lineage. These results seem to support the hypothesis of the evolution of the C. difficile 630 erm(B) determinant. The functional significance of one or two copies of erm(B) gene in C. difficile strains should be further investigated.     

TCV-309, a novel platelet activating factor antagonist,inhibits leukocyte accumulation and protects against splanchnic artery occlusion shock

The aim of this study was to evaluate: (1) the accumulation of leukocytes in the ileum and the lung during splanchnic artery occlusion (SAO) shock; (2) the role of platelet-activating factor (PAF) and tumor necrosis factor (TNF-) in this phenomenon. Untreated anesthetized rats subjected to total occlusion of the celiac, superior and inferior mesenteric arteries for 45 min, followed by reperfusion, uniformly died within 90 min after reperfusion. The mean survival time was 93±7 min. The neutrophilic infiltrate was quantitated in the ileum and in the lung using a myeloperoxidase (MPO) assay. MPO activity in the ileum and in the lung averaged 0.05±0.03 and 0.4±0.02 U×10^–3/g protein in animals killed before occlusion. MPO activity did not change in rats killed immediately before reperfusion and was significantly elevated (0.11±0.02 and 1.7±0.6 U×10^–3/g protein in the ileum and the lung, respectively) in those killed 80 min after the beginning of the reperfusion. The histological examination confirmed the accumulation of leukocytes in the mucosa of the ileum and the lung over the 80 min. SAO shocked rats exhibited leukopenia and increased serum levels of TNF-. In order to evaluate the role of PAF and TNF- in SAO shock, a powerful PAF receptor antagonist, TCV-309 (5 g/kg i.v.), was injected 5 min after reperfusion. TCV-309 increased survival time, lowered serum TNF-, reduced MPO activity in both the ileum and the lung and ameliorated leukopenia induced by SAO shock. In addition, the drug significantly reduced ileal necrosis and pulmonary morphological alterations induced by shock. These results suggest an important role for PAF in the adhesion of leukocytes in SAO shock.     

Cloning and characterization of two catechol 1,2-dioxygenase genes from Acinetobacter radioresistens S13

Two novel catechol 1,2-dioxygenase (C 1,2-O) genes have been isolated from an Acinetobacter radioresistens strain that grows on phenol or benzoate as sole carbon and energy source. Designated as catA(A) and catA(B), they encode proteins composed of 314 and 306 amino acids, whose deduced sequences indicate that they have approximately 53% identity, whereas their NH2-terminal and COOH-terminal regions have no sequences in common. This may explain their different thermal and pH stability. Polyclonal antibodies raised against an amino-terminal CatA(A) peptide or the whole CatA(B) protein were used to establish their inducible and differential expression patterns upon bacterial growth in phenol or benzoate. The CatA(A) protein (IsoA) was induced by both phenol and benzoate though with different kinetics, whereas the catA(B) product (IsoB) was constitutively produced at low levels that increased only during growth in the presence of benzoate.     

Biodegradation of poly(D(–)-3-hydroxybutyrate)/atactic poly(epichlorohydrin) blends by aureobacterium saperdae

The biodegradability of solution-cast films of poly(D (–)-3-hydroxybutyrate) (PHB) blended with the melt-compatible component atactic poly(epichlorohydrin) (aPECH) was investigated. A bacterium which produced extracellular enzymes that degraded PHB even when blended with aPECH was isolated, and tentatively designated as Aureobacterium saperdae. The growth rate of A. saperdae decreased with increasing aPECH content in the blend, up to films containing 60 wt.-% aPECH, at which composition growth was completely inhibited. The decrease in the bacterial growth rate could be due to the dilution of PHB molecules on the blend film surface caused by the presence of aPECH molecules. At the stationary phase of bacterial growth the percentage of weight loss of blend films decreased with increasing aPECH fraction, which was probably due to the lower accessibility of PHB when blended with aPECH. During the bacterial growth only PHB was metabolized, whereas neither degradation nor abiotic release of aPECH was detected for blend films.     

Candida albicans expresses a focal adhesion kinase-like protein that undergoes increased tyrosine phosphorylation upon yeast cell adhesion to vitronectin and the EA.hy 926 human endothelial cell line

The signaling pathways triggered by adherence of Candida albicans to the host cells or extracellular matrix are poorly understood. We provide here evidence in C. albicans yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain C. albicans yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited C. albicans yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of alpha v beta 3 and alpha v beta 5 integrin-like on C. albicans either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in C. albicans yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a C. albicans mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that C. albicans Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells.