Induction of human airway smooth muscle apoptosis by neutrophils and neutrophil elastase

Neutrophils are an important component of airway inflammation and may interact with human airway smooth muscle cells (HASMC). We investigated the effect of neutrophils and of neutrophil-derived proteases on HASMC survival. When co-incubated with neutrophils (0.1-1 x 10(6) cells/ml), attachment of human ASMC was reduced to 12.3 +/- 4.3% compared with untreated controls after 72 h. HASMC showed nuclear condensation and fragmentation (41.6 +/- 8.1% compared with baseline of 3.1 +/- 0.4%), and the biochemical markers of apoptosis, annexin V binding (9.7 +/- 0.7%; baseline 1.1 +/- 0.3%) and cleaved caspase-3 expression, were observed. The proteolytic activity released by neutrophils was essential for the proapoptotic effect because inhibition of elastase activity by alpha(1)-antitrypsin and MeOSuc-Ala-Ala-Pro-Ala-CMK (MSACK) reduced HASMC apoptosis. Human neutrophil elastase (0.1-3 microg/ml) induced apoptosis of HASMC, as well as other neutrophil serine proteases, cathepsin G, and proteinase 3. Fibronectin degradation products were present in HASMC supernatants exposed to neutrophil-conditioned media and to neutrophil elastase. The local release of proteases from neutrophils present in airway smooth muscle cells may lead to HASMC apoptosis as a result of matrix degradation and loss of cell attachment. This may limit pathologic changes such as ASMC hyperplasia and extracellular matrix deposition seen in airway remodeling.     

Lung preservation solution substrate composition affects rat lung oxidative metabolism during hypothermic storage

Lungs harvested for transplantation utilize oxygen after procurement. We investigated the effects of storage solution substrate composition on pulmonary oxidative metabolism and energetics during the preservation interval. Rat lungs were harvested and stored at 10 degrees C in low-potassium dextran (LPD) solution. Groups of lungs were preserved with preservation solution containing 5mM carbon-13 ((13)C) labeled glucose or increasing concentrations of (13)C labeled pyruvate. Additional groups of rat lungs were studied with dichloroacetate (DCA) added to the pyruvate-modified preservation solutions. Oxidative metabolism (measured by (13)C-enrichment of glutamate) and adenine nucleotide levels were quantified. Increasing preservation solution pyruvate concentration augmented glutamate (13)C-enrichment up to a concentration of 32mM pyruvate. DCA further stimulated oxidative metabolism only at lower concentrations of pyruvate (4 and 8mM). ATP and ADP were not different among groups, but AMP levels were higher in the glucose group. These data suggest that altering the substrate composition of the preservation solution influences lung metabolism during allograft preservation for transplantation.     

ELAV inhibits 3'-end processing to promote neural splicing of ewg pre-mRNA

The embryonic lethal abnormal visual system (ELAV) is a gene-specific regulator of alternative pre-mRNA processing in neurons of Drosophila. Here we define a functional in vivo binding site for ELAV in neurons through the development of a reporter gene system in transgenic animals in combination with in vitro binding assays. ELAV binds to erect wing (ewg) RNA 3' of a polyadenylation site in the terminal intron 6. At this polyadenylation site, ELAV inhibits 3'-end processing in vitro in a dose-dependent and sequence-specific manner, and ELAV binding is necessary in vivo to promote splicing of ewg intron 6. Further, the AAUAAA poly(A) complex recognition sequence, together with ELAV, is required to regulate neural 3' splice site choice in vivo. In addition, the use of segmentally labeled RNA substrates in UV cross-linking assays suggest that ELAV does not inhibit or redirect binding of cleavage factor dCstF64 at the regulated polyadenylation site on ewg RNA. These data indicate that binding of 3'-end processing factors, together with ELAV, can regulate alternative splicing.     

Two sisters with Escobar syndrome

We report on 2 sisters with an autosomal-recessive multiple pterygium syndrome, type Escobar, consisting of multiple pterygia with severe contractures, short stature, and minor facial and external genital anomalies. The striking finding was severe muscular atrophy. We speculate that a neu-romuscular disorder is the underlying pathogenesis of Escobar syndrome. © 1995 Wiley-Liss, Inc.     

Postoperative Paenibacillus thiaminolyticus Wound Infection,Switzerland

Paenibacillus thiaminolyticus is a nonvirulent organism found in human and ruminant microbiota. However, P. thiaminolyticus can act as an opportunistic pathogen in humans. We describe a case of abdominal wall hematoma secondarily infected by P. thiaminolyticus. Our findings emphasize the risk for unusual Paenibacillus infections in otherwise healthy persons.     

Analysis of odontoblast gene expression using a novel approach,laser capture microdissection

Studying the mechanisms of molecular interactions in developing tissues demands sensitive molecular biological in vivo and in vitro techniques. Laser capture microdissection (LCM) allows for the isolation of mRNA in histological sections even from single cells, thus enabling the identification of in vivo gene expression products in closely circumscribed tissue areas. The aims of this study were to assess the optimal fixation, processing, and staining conditions to retrieve RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of alpha 1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA isolated from day 0 and day 3 captured odontoblasts was sufficient in quantity and quality. Our results indicate that individual odontoblasts obtained by LCM are morphologically intact and chemically unaltered, allowing accurate molecular and biochemical analyses.     

Large-field S-cone flicker test

Background: Disturbances of blue color vision and of temporal contrast sensitivity can indicate early damage in glaucoma. For the present study a quick and easy test was devised which examines both functions ai one time by testing the temporal contrast sensitivity of a blue flickering light on an intense yellow background. Methods: Large coextensive background and test fields (85°) are used, making fixation uncritical. Detailed experiments were made in two normal subjects to derive spectral sensitivity curves from flicker-fusion frequency (FFF) versus intensity functions and to obtain complete temporal contrast-sensitivity (De Lange) curves under different levels of adaptation and test lights. After selection of appropriate luminances and one stimulation frequency from these experiments, test-retest variability was studied in four subjects in five repetitions. In addition, normal values were collected from 22 subjects. Results: Spectral sensitivities for two levels of FFF (15 Hz and 44 Hz) agree with Stiles' ₁ at the low and with ₄ at the high FFF. Temporal contrast-sensitivity curves show a low-frequency section with peak sensitivity at 1 Hz and a high-frequency section with a peak at around 4 Hz. From the basic experiments the following conditions for the clinical examination were selected: Background luminance 2600 cd/m², test luminance at 451 nm 0.8 cd/m², stimulation frequency 4 Hz. The test-retest variability showed an acceptable intraclass correlation coefficient (0.6). Conclusions: The present experiments carried out with a very large stimulus led to meaningful results which are in rather good agreement with results reported in the literature on small-field stimuli. The blue-on-yellow flicker test carried out under the conditions mentioned above is a quick and easy test which could be helpful in improving early glaucoma diagnosis.