Pseudomonas pyocyanin increases interleukin-8 expression by human airway epithelial cells

Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1alpha. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease.     

Prolonged administration of NT-4/5 fails to rescue most axotomized retinal ganglion cells in adult rats

The survival of axotomized RGCs was increased by intravitreal NT-4/5 given by repeated injections or osmotic minipumps, but the effects were less complete than predicted. Compared to a single injection of the neurotrophin on day 0, second injections on days 3 or 7 only sustained an additional 10-20% of the RGCs on day 10. Minipumps augmented RGC survival up to 4-fold (50%) at 2 weeks but most RGCs were lost by 1 month. Thus, specific neurotrophins can rescue many RGCs soon after injury but long-term neuronal survival may require a better understanding of changes in neurotrophin receptors and interactions with other molecules.     

Partial depletion and multiple deletions of muscle mtDNA in familial MNGIE syndrome

OBJECTIVE: To describe the unique combination of partial depletion and multiple deletions of mitochondrial DNA (mtDNA) on muscle DNA analysis of three siblings with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). BACKGROUND: MNGIE is a relatively homogeneous autosomal recessive disorder characterized by gastrointestinal dysmobility, ophthalmoparesis, peripheral neuropathy, mitochondrial myopathy, and altered white matter signal at brain imaging. Muscle multiple mtDNA deletions have been found in about half of the described cases. METHODS: We studied three affected siblings (two were monozygotic twins) born to nonconsanguineous parents. Muscle mtDNA was investigated by quantitative Southern and Slot blot techniques and by PCR analysis. Morphologic confirmation in the muscle tissue was achieved by using in situ hybridization with a mtDNA probe complementary to an undeleted region and by DNA immunohistochemistry. RESULTS: All three patients showed ragged red (RRF) and cytochrome c oxidase-negative fibers, as well as partial deficiency of complexes I and IV. Southern and Slot blot analyses showed mtDNA depletion in all patients. Multiple mtDNA deletions were also detected by PCR analysis. In situ hybridization demonstrated an overall signal weaker than controls, with a relatively higher signal in RRF. Antibodies against DNA showed a decreased cytoplasmic network. CONCLUSIONS: The muscle histopathology and respiratory chain enzyme defects may be accounted for by the decreased mtDNA amount and by the presence of mtDNA deleted molecules; however, relative levels of mtDNA seem to correlate with life span in these patients. The combination of partial depletion and multiple deletions of mtDNA might indicate the derangement of a common genetic mechanism controlling mtDNA copy number and integrity.     

Disease and sleep satisfaction at age 70

A new arylnaphthalene lignan, 9-(1,3-benzodioxol-5-yl)-4,5,6, 7-tetramethoxynaphtho[2,3-C]furan-1(3H)-one (5-methoxyjusticidin A, 1), was isolated from a Et2O extract of the wood of Protium unifoliolatum. The structure of 1 was determined by both spectroscopic and X-ray crystallographic methods.     

Stimulation of human cytotoxic T cells with HIV-1-derived peptides presented by recombinant HLA-A2 peptide complexes

HLA-A2 heavy chain and beta 2-microglobulin were expressed in Escherichia coli, and refolded in the presence of peptides derived from HIV-1 RT and gag proteins. When recombinant HLA-A2 molecules were attached to cells lacking HLA-A2, the cells became susceptible to lysis by HLA-A2-restricted cytotoxic T lymphocyte (CTL) clones specific for peptides derived from RT and gag proteins. Limiting dilution analyses of peripheral blood mononuclear cells from HIV-1-infected individuals showed that the recombinant HLA-A2 peptide complexes covalently immobilized on microspheres stimulated the development of HLA-A2 peptide-specific CTL. Preformed HLA-peptide complexes may provide an alternative to immunization procedures that depend upon intracellular processing of antigen to elicit T cell responses.     

Correlation of heat resistance and HSP-70A mRNA levels in human tumor cells measured by competitive quantitative polymerase chain reaction

PURPOSE: Several HSP-70 genes have been cloned and sequenced in human cells. Among these genes, the HSP-70A mRNA and protein levels correlate best with the development and decay of thermotolerance and intrinsic thermal sensitivity. Leukemic and nonleukemic human tumor cells express low levels of the normally heat inducible HSP-70A mRNA in control nonheated cells. Using a competitive quantitative polymerase chain reaction, we have measured the levels of mRNA for this gene and have correlated it with both transient and intrinsic thermal sensitivity of tumor cells. Such studies were also extended to tumor samples obtained from patients. METHODS AND MATERIALS: In these studies, the plasmid phHSP-70 which contains the entire human HSP-70A gene was modified by the insertion of the T7 promoter at the 5'-end untranslated region as well as the insertion of a 23 bp synthetic linker at the BamH1 site in the promoter region of the HSP-70A gene. The PCR primers were located such that the amplified fragment contained the linker. Using the T7 polymerase, the HSP-70A mRNA was transcribed from this plasmid (phHSP-70L) in vitro. A known amount of HSP-70A mRNA was then added to the total RNA prepared from the cell samples or from the tumor tissues obtained from patients. Using the components of the PCR reaction plus known amounts of HSP-70A mRNA synthesized from phHSP-70L and unknown amounts of total cellular RNA, the samples were amplified and analysed on a denaturing acrylamide gel. The PCR products obtained from phHSP-70L were 23 bp larger than the PCR products obtained from the cell samples due to the addition of the synthetic linker to the HSP-70A gene in phHSP-70L and therefore, the two products could be easily distinguished from each other and quantitated. The alpha-32P-dCTP incorporated in each sample was quantitated by AMBIS Scanner. When the 32P-counts were equal in the known and the unknown samples, the amount of the HSP-70A mRNA was taken to be equal in the known and the unknown sample. RESULTS: The results show that HSP-70A mRNA levels can be used to predict the survival levels during the development and decay of thermotolerance. In nonleukemic human tumor cell lines, there are as much as 40-50-fold induction of HSP-70A mRNA levels during the peak of thermotolerance. In leukemic cell lines, however, HSP-70A mRNA levels are induced only by three-fold during the same time period. These differences between the levels of HSP-70A mRNA positively correlate with the amount of tolerance development in leukemic and nonleukemic tumor cells. HSP-70A mRNA levels also vary in different tumor cells under nonheated conditions and there is a positive correlation between HSP-70A mRNA levels in nonleukemic human tumor cells and the level of their intrinsic thermal resistance. CONCLUSION: HSP-70A mRNA levels can be used to predict the intrinsic thermal sensitivity of nonleukemic human tumor cells.