Sputum neutrophils in cystic fibrosis patients display a reduced respiratory burst.

BACKGROUND: Few data exist on the functional activity of airway neutrophils in the milieu of the cystic fibrosis (CF) lung. We assessed reactive oxygen species (ROS) production by sputum neutrophils and the relationship to neutrophil viability. Identical assessments were made on peripheral blood neutrophils from CF patients. METHODS: ROS production in sputum neutrophils was assessed in 31 CF patients at varying phases of clinical disease using flow cytometry. Twenty patients provided blood samples (including 16 who also provided a matched sputum sample). Neutrophil viability was determined using dual annexin V (apoptosis) and propidium iodide (necrosis) staining. Comparative peripheral blood data were obtained from 7 healthy controls. RESULTS: ROS production was reduced in sputum compared to blood neutrophils and they demonstrated a higher level of necrosis. Subpopulations of neutrophils with different ROS production capacity were apparent in peripheral blood. Lung function was positively associated with both the proportion of blood neutrophils demonstrating increased ROS production and the proportion of apoptotic sputum neutrophils. CONCLUSIONS: CF airway neutrophils display functional exhaustion. Healthier lungs in CF appear to be associated with subpopulations of blood neutrophils with increased oxidative burst capacity and evidence for increased neutrophil apoptosis within the airway.

The effect of herbal medicines on platelet function: an in vivo experiment and review of the literature.

BACKGROUND: Herbal medicines are used by a considerable number of surgical patients. An increased risk of bleeding, substantiated by anecdotal reports, has been attributed to the use of certain herbs, and numerous in vitro experiments have identified some herbal extracts as platelet inhibitors. The purpose of this investigation was to determine whether standard commercial preparations of commonly used herbal medicines have an effect on platelet function in vivo and, by extension, to provide clinical scientific evidence of the safety of their use in the perioperative period. METHODS: Five commercially available herbal agents were investigated, including Ginkgo biloba, garlic, Asian ginseng, St. John's wort, and saw palmetto. In a blinded fashion, one of the agents was administered to 10 adult volunteers at the manufacturer's recommended dose for 2 weeks. At the end of the 2-week period, in vivo platelet function was quantified using the PFA-100 assay. After a 2-week "washout" period, the protocol was repeated using a different agent. This 4-week cycle was repeated for each of the five herbal agents, as well as the control agent aspirin. RESULTS: In vivo platelet function was not affected by the administration of any herbal agent and was markedly inhibited with the administration of aspirin. CONCLUSIONS: The herbal medicines investigated in this study do not affect platelet function in vivo. Neither this experiment nor a review of the literature supports the concern of perioperative bleeding in users of these herbal medicines.

Blood group B degrading activity of Ruminococcus gnavus alpha-galactosidase.

Ruminococcus gnavus is a Gram positive, nonspore-forming obligate anaerobe normally found in the human alimentary tract. In culture, this organism constitutively produces a 1-3 alpha-galactosidase. We fractionated and characterized this enzyme demonstrating hydrolysis of the B epitope on erythrocyte membranes and seroconversion to H epitope (blood type O). Since the enzyme yield was low, cell suspension studies could not be performed. Instead, hydrolysis of the B membrane epitope was studied with an ELISA. A highly purified enzyme product was analyzed for characteristics such as pH, ionic strength, and temperature optimum. Activity in red cell preservative solutions and in the presence of type B plasma was also demonstrated. Ruminococcus gnavus a 1-3 alpha-galactosidase has potential application in the enzymatic conversion of type B to O packed red blood cell units.

Purification and characterization of a recombinant alpha-N-acetylgalactosaminidase from Clostridium perfringens.

Clostridium perfringens alpha-N-acetylgalactosaminidase (alphaNAG) hydrolyzed the terminal N-acetyl-alpha-d-galactosamine from the blood type A(2) antigen producing H antigen, blood type O. Blood type O is universally compatible in the ABO system. Purification of the native enzyme is difficult with very low yields. To obtain the enzyme in satisfactory yield, the gene encoding the clostridial enzyme was cloned in an Escherichia coli T7 expression system. A highly purified preparation of recombinant alphaNAG was obtained from cell lysates by ion-exchange chromatography and high-pressure liquid chromatography. The final preparation was homogeneous by SDS-PAGE with a molecular mass of 71.96kDa and the native molecular weight of 72.42kDa. The enzyme was highly selective for terminal N-acetylgalactosamine residues. No other significant exoglycosidase activities, particularly neuraminidase, were detected. The pH optimum of the enzyme was between 6.5 and 7.0 and activity was relatively unaffected by ionic strength. ELISA experiments demonstrated activity against blood type A(2) epitope. These characteristics were similar to those of native alphaNAG from C. perfringens. With adequate expression in E. coli, sufficient recombinant alphaNAG enzyme mass can be obtained for potential use in enzymatic conversion of human blood type A(2) red blood cells to universally transfusable type O red blood cells.

Identification, molecular cloning and expression of an alpha-N-acetylgalactosaminidase gene from Clostridium perfringens.

The Clostridium perfringens gene encoding the previously characterized alpha-N-acetylgalactosaminidase (alphaNAG) was identified by protein microsequencing and database searching. The alphaNAG protein, designated AagA, was found to be encoded by a hypothetical gene of unknown function in the recently completed genome sequence of C. perfringens strain 13. The deduced translation product of 629 amino acid residues possessed a region of limited homology to several hypothetical open reading frames, an enterotoxin of unknown function and several known or predicted alpha-galactosidases, but did not exhibit homology to any of the multiple sequenced eukaryotic alphaNAG proteins. The C. perfringens aagA gene, encoding AagA, was cloned in an Escherichia coli T7 expression system, resulting in recombinants exhibiting high-level expression of the expected alphaNAG activity. To our knowledge, this is the first report of the cloning and expression of a bacterial alphaNAG-encoding gene and represents an important step in the development of recombinant alphaNAG as a tool in the enzymatic conversion of blood group antigens.