Iloprost in alginate decreases the thrombogenicity of expanded polytetrafluoroethylene.

Vascular prostheses of small diameter perform poorly in vivo, in part because of the high thrombogenicity of available biomaterials. This study examined the thrombogenicity of expanded polytetrafluoroethylene (ePTFE) vascular graft segments (10 mm long x 4 mm i.d.) in vitro before and after impregnation with saline, alginate, or alginate containing the stable prostacyclin analog, iloprost. Each segment was immersed in activated whole blood and the weight of the adherent thrombus was measured at specified intervals. At 6 and 7 min the saline-denucleated group accumulated significantly less thrombus than control (p < .05). Alginate alone was not significantly different from controls. The graft segments treated with alginate + iloprost accumulated significantly less thrombus (p < .05) than all other groups after 6 min. These data demonstrate that denucleation of ePTFE with iloprost in alginate dramatically decreases its in vitro thrombogenicity.

Outcome of amputations in patients with major burns.

There is little mention in the literature of the outcome of patients who require amputations for large surface area burns. To determine their outcome, we devised a case-control study. Fifteen patients who underwent amputations between 1984 and 1993 at the University of Louisville Burn Unit were analysed and compared to a similarly injured control group. Both groups represented severely burned patients with high total body surface areas involvement. Apache II scores, and per cent of inhalation injuries. The results showed a 60 per cent survival rate in each group. Unlike previous reports on electrical burns, the amputations in this series of primarily thermal injuries (12 of 15 patients) were performed late in the hospital course (mean, 15 days) and after previous attempts at limb salvage (mean, two procedures). By eliminating either non-viable or infected tissue, amputations served a role in obtaining a respectable survival rate for these severely injured patients that also compared favourably with the control group.

Identification and initial characterization of Pneumocystis carinii soluble antigens in rabbit serum and lung lavage.

Lung lavage and serum samples from Azathioprin-treated (acute-phase infection) and untreated (non acute-phase infection) rabbits were used in the immunoblotting technique to look for Pneumocystis carinii (Pc) soluble antigens, using rabbit polyclonal antibodies raised against rabbit-derived Pc antigens and labeled with peroxidase. Analysis of the supernatant of lavage fluid after centrifugation to sediment intact organisms revealed components of approximately 80, 60-65, 55, 39 and 27 kDa in acute-phase samples. The components in the regions of 80, 60-65, 55 kDa and to a lesser extent 39 kDa were also present in non acute-phase lung lavage samples. In acute-phase serum samples, a major component of 80 kDa and minor components of about 65 and 39 kDa are detectable. The 80 and 65 kDa components are also detectable in some of the serum samples from the untreated rabbits. Immunofluorescent staining with FITC-conjugated affinity-purified antibodies to the 80, 60-65, 55, 39 or 27 kDa-components showed that they shared epitopes with both Pc cysts and trophozoites. The affinity-purified antibodies also cross-reacted in immunoblotting with several antigens in the Pc whole preparations. The putative Pc soluble antigens in serum and lung lavage were then isolated by affinity chromatography with polyclonal antibodies to Pc. Preliminary characterization of the column-extracted antigens revealed complete inactivation by trypsin whereas only the 55 and 80 kDa antigens bind to Concanavalin A. In conclusion, the results of this study suggest that Pc soluble antigens are present even in non acute-phase samples and only the low-molecular weight antigens (39 and 27 kDa) seem specific for the acute-phase. These findings are consistent with previous investigations reported by others that development of Pc could occur in nonimmu-nosuppressed rabbits.

Production and characterization of monoclonal antibodies to human Pneumocystis carinii for the diagnosis of P. carinii pneumonia.

OBJECTIVES: Monoclonal antibodies against human Pneumocystis carinii were produced by fusion of myeloma cells (X63-AG8.653) with splenocytes from Biozzi mice that had been immunized against P. carinii cysts isolated from infected human lung. The aim of this study was to evaluate the usefulness of these monoclonal antibodies for the diagnosis of P. carinii pneumonia by indirect immunofluorescence in comparison with a modified silver stain method and commercial kits. METHODS: One hundred fifty-seven specimens from 87 patients, infected or non-infected with human immunodeficiency virus, were examined for the presence of P. carinii. Specimens were either induced sputum samples or bronchoalveolar lavage fluids. Indirect immunofluorescence was performed with six stable clones obtained by limiting dilution. Four of the monoclonal antibodies were IgG1 isotypes, one was an IgG3 and one was an IgM. Their isoelectric points varied from 6.5 to 8.3. Tests were also performed with silver methenamine staining and with two commercially available monoclonal antibodies (Monofluo kit from Diagnostics Pasteur and MAb from Dako). RESULTS: The 6 antibodies all recognized P. carinii cysts in indirect immunofluorescence. No cross reactivity was observed with yeast or host cells. P. carinii antigens could not be identified with western immunoblotting suggesting that the monoclonal antibodies recognized native antigens. This result was confirmed by dot blot analysis. Spots were observed with native but not with denatured antigens. Inhibition studies showed that these 6 antibodies recognized the same or overlapping sites. The sensitivities of detection of P. carinii in sputum were 87% by silver stain and from 93.5 to 96.7% by immunofluorescence. The sensitivities of detection in bronchoalveolar lavage were 67.3% by silver stain and from 75.7% to 76.8% by immunofluorescence. CONCLUSION: Immunofluorescence was more sensitive than silver staining and the best results were obtained with E5-8 and A8-13 monoclonal antibodies and with Monofluo kit.

Analysis of Pneumocystis carinii antigens by CIEP, SDS-PAGE and immunoblotting.

Pneumocystis carinii (Pc) from rabbit and rat lungs were analyzed by counterimmunoelectro-phoresis (CIEP), Polyacrylamide gel electrophoresis and immunoblotting. In CIEP, considerable cross-reactivity was demonstrated using rabbit polyclonal antisera raised against the rabbit and the rat-derived Pc components. In immunoblotting, both the rat and the rabbit Pc antigens also showed reactivity with the two antisera. Rabbit-derived Pc antigens of about 24-27,39,42-45, 55, 110 and 116 kDa were revealed with infection-derived rabbit sera. The 39, 42-45 and 116 kDa antigens were further recognized by both human and rat immune sera. In addition, many human sera detected the rabbit Pc antigen of 42-45 kDa. Using rat infection-derived sera, rat Pc antigens of 39, 55 to 60, 110 and 116 kDa were identified. The sera from Pc infected rabbits and humans recognized the bands in the regions of 39, 55 and 116 kDa. Carbohydrate residues were further analyzed by Concanavalin A (Con A)-binding experiments, periodate Schiff (PAS) and Alcian Blue (AB) stainings. The 55 and 116 kDa bands of both rabbit and rat Pc strongly reacted with Con A and were stained by PAS indicating the presence of hydroxyl and mannosyl/glycosyl residues. The AB staining-bands were 39, 42-45 and 55 kDa for rabbit Pc isolates and 39, 45, 55-60 and 70 kDa for rat Pc isolates, indicating the presence of carbohydrate residues with acidic function. The rabbit Pc immunodominant 39, 55 and 116 kDa antigens then appeared to be glycoproteins with characteristics similar to those of their counterparts in rats. The implications of these findings are discussed.