Production of serine chymotrypsin-like elastate by aspergillus fumigatus strains

Treinta y cuatro cepas de Aspergillus fumigatus aisladas del aire, crín de caballo, suelo agrícola y del hombre, fueron examinadas con el fin de evaluar la producción de elastasa. Las cepas de Aspergillus fumigatus fueron cultivadas en un medio sólido con elastina, apreciándose en ella su amplio solubilización por la acción del hongo. Los aislamientos fúngicos provenientes de muestras aisladas del hobre y de suelos agrícolas fueron detectados como los más altos productores de elastasa. Ocho de las 34 cepas fueron desarrollas en 4 diferentes medios líquidos en las cuales se investigó la actividad proteolítica total y específica. Los resultados de este experimento sugieren que la producción de elastasa es inducida por la presencia de elastina como sustrato y que la primera es una enzima semejante a la quimiotripsina. El perfil inhibitorio comprobó que la elastina de A.fumigatus, es una serina-proteinasa

Non specific immunity in aged healthy subjects and in patients with chronic bronchitis.

We investigated the effects of aging on some functional activities (chemotaxis, phagocytosis, nitroblue tetrazolium reduction and candidacidal activity) of peripheral polymorphonuclear and mononuclear phagocytes in 96 healthy subjects and 89 patients with chronic bronchitis, aged 40 to 100 years. The subjects were divided according to age into younger (40-65 years) and older (66-100 years) individuals. No subject was taking any drug known to affect phagocytic functions. A few abnormalities in PMN and monocyte functions were observed in aged healthy subjects, in comparison to the younger ones; in fact, only the chemotactic response to complement-derived chemotactic factors was significantly impaired in elderly healthy individuals. On the contrary, multiple alterations of phagocyte activities, i.e., chemotaxis, phagocytosis, and candidacidal activity were observed in aged subjects with chronic bronchitis, compared to healthy adults. However, the results obtained in older and younger patients with chronic bronchitis were superimposable. The present data suggest that the decline in functional activities of phagocytes in the aged could depend on the effect of the underlying chronic bronchitis on the cellular components of the non-specific host defense system, rather than a direct effect of the aging process.

Proteinase/proteinase inhibitor imbalance in sputum sol phases from patients with chronic obstructive pulmonary disease. Suggestions for a key role played by antileukoprotease.

In order to characterize the imbalance between proteinases and proteinase inhibitors in sputum sol phases, we studied 25 patients (mean age, 59 +/- 11 yr) with exacerbated chronic obstructive pulmonary disease (COPD). An aliquot of sputum was used for bacteriologic determinations, and the remainder was centrifuged in order to obtain gel and sol phases. On the basis of the bacteriologic data, patients were divided into colonized patients (14) and noncolonized patients (11). All of the major inhibitors were immunologically detectable in sol phases without a significant difference between colonized and noncolonized patients (alpha 1-proteinase inhibitor [alpha 1-PI], 2.56 microM +/- 0.53 microM and 2.39 microM +/- 0.72 microM; alpha 2-macroglobulin [alpha 2-MG], 0.21 microM +/- 0.07 microM and 0.16 microM +/- 0.05 microM; antileukoprotease (ALP), 1.78 microM +/- 0.57 microM and 1.53 microM +/- 0.6 microM, respectively [mean +/- SE]). With regard to proteinase activities, both free elastase-like and free chymotrypsin-like activities were detectable in the majority of patients (15/25) (0.59 microM +/- 0.15 microM and 0.74 microM +/- 0.15 microM for elastase-like activity [ELA], and 0.010 microM +/- 0.003 microM and 0.017 microM +/- 0.007 microM for chymotrypsin-like activity [CLA], respectively [mean +/- SE]). The inhibitory profile of proteinase activities, performed by means of a panel of inhibitors, allowed us to assign specific activities mainly to neutrophil elastase and cathepsin G (Cat G). Next we looked at the relationships between inhibitors and proteinase activities. We found a significant negative correlation between neutrophil elastase activity and ALP (r = -0.58; p < 0.01). In confirmation of this suggestion, sol phases were divided into samples (15) with detectable ELA (> 0.50 microM) and samples (10) with no detectable ELA (< 0.18 microM). Levels of alpha 1-PI and alpha 2-MG did not differ significantly between the two groups, whereas ALP values were higher in the group with no detectable ELA (3.12 microM +/- 0.69 microM) than in the other group (0.58 microM +/- 0.21 microM; p < 0.001). We conclude that most sputum sol phases from patients with exacerbated COPD have a high burden of free neutrophil elastase and Cat G. Antileukoprotease seems to be the major naturally occurring inhibitor effective in the modulation of proteinase activities in bronchial secretions under these conditions.

Protease-antiprotease imbalance: local evaluation with bronchoalveolar lavage.

The protease-antiprotease imbalance is thought to be involved in a variety of destructive lung diseases: pulmonary emphysema, chronic bronchitis, cystic fibrosis and adult respiratory distress syndrome. Bronchoalveolar lavage allowed the investigators to assess the protease-antiprotease shift in such conditions but sometimes gave conflicting results. The role of bronchoalveolar lavage as a research and diagnostic tool in diseases characterised by protease-antiprotease imbalance is reviewed, as well as its potential usefulness in the near future.

Some properties of the alkaline proteinase from Aspergillus melleus.

Seaprose is a semi-alkaline proteinase produced by Aspergillus melleus. The aim of our study was to further characterize the properties of this enzyme, particularly looking at its interaction with alpha 1-proteinase inhibitor, the major human plasma proteinase inhibitor. We studied the cleavage of three synthetic peptide substrates induced by seaprose and the inhibitory profile of the enzyme by means of a panel of inhibitors, including alpha 1-proteinase inhibitor. The interaction between seaprose and alpha 1-proteinase inhibitor was also studied with SDS-PAGE. Finally, the elastolytic activity of seaprose was checked by means of bovine elastin solubilization. We found that seaprose cleaves preferentially the substrate containing a Phe residue in the P1 position. The inhibitory profile showed that seaprose is a serine-proteinase that cannot be inhibited by alpha 1-proteinase inhibitor. The SDS-PAGE revealed that alpha 1-proteinase inhibitor, after incubation with seaprose, underwent a limited proteolysis. Finally, seaprose 10(-2) M and 10(-3) M was able to solubilize bovine elastin. We conclude that seaprose is a serine-proteinase able to inactivate human alpha 1-proteinase inhibitor with limited proteolysis at (or near) the active site and that it has mild elastinolytic capacity.

Alpha 1-antitrypsin variants produced by recombinant DNA: differences in elastase inhibitory activity and resistance to oxidant agents.

Inherited or "acquired" deficiency of alpha 1-antitrypsin (believed to be the cause of pulmonary emphysema) will probably be treated in the future by replacement with alpha 1-antitrypsin purified from human plasma or produced by recombinant DNA, which seems promising because it permits site-specific mutagenesis in the oxidizable active site of the normal human alpha 1-antitrypsin. The aim of this in-vitro study was to investigate the elastase inhibitory activity and the resistance to oxidizing agents of normal human alpha 1-antitrypsin, a recombinant yeast-produced variant (VAL 358) and a recombinant E. coli-produced variant (LEU 358). The inhibitors were exposed to chemical oxidants (NCS, H2O2, xanthine/xanthine oxidase, chloramine-T) and to PMA-activated neutrophils. The elastase inhibitory activity was assayed on porcine pancreatic elastase and neutrophil elastase. Normal alpha 1-antitrypsin and VAL 358 variant were good inhibitors of both elastases. LEU 358 variant was the best inhibitor for neutrophil elastase, but it poorly inhibited the porcine pancreatic elastase. Normal alpha 1-antitrypsin was affected by all oxidants; both variants were almost totally resistant to chemical oxidants and to activated neutrophils. We conclude that recombinant alpha 1-antitrypsin variants differ in their elastase inhibitory activity and offer increased resistance to oxidant agents.

Studies of MR 889, a new synthetic proteinase inhibitor.

We investigated the proteinase inhibitory activity of MR 889, a thiolactic acid derivative. It is able to in vitro inhibit at low concentration (10(-5),10(-6)M) the activity of porcine pancreatic elastase, human neutrophil elastase and bovine chymotrypsin. In addition, MR 889 is able to inhibit the residual activity of alpha 2-macroglobulin-trapped human neutrophil elastase, paralleling the efficacy of phenylmethylsufonylfluoride. Finally, MR 889 has been shown to in vitro reduce the burden of elastase- and chymotrypsin-like activity found in sputum sol-phases of patients admitted for chronic bronchitis exacerbation.

Bactericidal activity of meropenem and interactions with other antibiotics.

MICs of meropenem for selected clinical isolates of bacteria were determined. Killing curves were performed on strains of methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staph. aureus (MRSA), methicillin-resistant Staph, epidermidis, Escherichia coli, Klebsiella spp., Enterobacter cloacae, Pseudomonas aeruginosa, Citrobacter freundii and Acinetobacter spp. A reduction of greater than or equal to 3 x log10 in viable cells was observed at 4 and 6 h of exposure to 4 and 8 x MIC, and this was usually maintained at 24 h (with a few exceptions for methicillin-resistant Staph, epidermidis and Ent. cloacae). At the MIC and twice the MIC regrowth tended to occur at 24 h although this varied from strain to strain. The interaction with other antibiotics was determined by the chequerboard technique. Usually an additive or synergistic effect was observed when meropenem or imipenem was used in combination with an aminoglycoside against Gram-negative species, while in a few cases antagonism occurred in combination with beta-lactams. Against Staph, aureus, MRSA and Staph, epidermidis synergism was usually obtained with combinations with teicoplanin or vancomycin and either synergism or addition with combinations with rifampicin, co-trimoxazole or ciprofloxacin.

[In vitro antibacterial activity of neomycin, bacitracin and a combination of both]. / Attività antibatterica "in vitro" di neomicina e bacitracina e della loro associazione.

Bacitracin and neomycin have been used for a long time for local intestinal antisepsis or decontamination, due to their scarce or nonexistent intestinal absorption. The aim of the present study was to determine the activity of bacitracin and neomycin against recent clinical isolates of aerobic and anaerobic microorganisms and to verify their capacity to have a synergistic effect and to prevent the emergence of bacterial resistance when in combination. The results showed that the activity of either antibiotic against recent clinical isolates (even if resistant to cephalosporins and aminoglycosides) is similar to that originally displayed at the time introduction in therapy. Generally the combination of the two antibiotics showed a synergistic or additive effect and prevented the selection of resistant strains.

[Ciprofloxacin by the intravenous route in the treatment of respiratory infections]. / La ciprofloxacina per via endovenosa nel trattamento delle infezioni respiratorie.

A clinical trial was performed to evaluate the efficacy of ciprofloxacin by iv administration in the treatment of respiratory infections. Twenty-two in-patients affected with acute lower respiratory tract infections, mainly infectious exacerbations of chronic obstructive lung disease (COLD), were treated with ciprofloxacin at the daily dosage of 400 mg iv, in two administrations. Overall clinical results were satisfactory (15 patients cured, 7 patients improved). Concerning bacteriological results, pathogen eradication was achieved in 83.3% of cases. Tolerability was good: no adverse events were observed.