DNA fingerprinting by ERIC-PCR for comparing Listeria spp. strains isolated from different sources in San Luis, Argentina.

In this study, a total of 24 Listeria spp. strains were analyzed. Twenty-two isolates were obtained in San Luis (Argentina) from human, animal, and food samples. Two types of strains, Listeria monocytogenes CLIP 22762 and Listeria innocua CLIP 74915, were included as reference strains. All isolates were biochemically identified and characterized by serotyping, phage typing, and amplification of the flaA gene by polymerase chain reaction (PCR). Repetitive intergenic consensus (ERIC) sequence-based PCR was used to generate DNA fingerprints. On the basis of ERIC-PCR fingerprints, Listeria spp. strains were divided into three major clusters matching origin of isolation. ERIC-PCR fingerprints of human and animal isolates were different from those of food isolates. In addition, groups I and II included ten L. monocytogenes strains, and only one Listeria seeligeri strain. Group III included nine L. innocua strains and four L. monocytogenes strains. Computer evaluation of ERIC-PCR fingerprints allowed discrimination between the tested serotypes 1/2b, 4b, 6a, and 6b within each major cluster. The index of discrimination calculated was 0.94. This study suggests that the ERIC-PCR technique provides an alternative method for the identification of Listeria species and the discrimination of strains within one species.

DNA fingerprinting by ERIC-PCR for comparing Listeria spp. strains isolated from different sources in San Luis: Argentina / Caracterización molecular por ERIC-PCR de cepas de Listeria spp. aisladas de diversos orígenes en San Luis: Argentina

In this study, a total of 24 Listeria spp. strains were analyzed. Twenty-two isolates were obtained in San Luis (Argentina) from human, animal, and food samples. Two types of strains, Listeria monocytogenes CLIP 22762 and Listeria innocua CLIP 74915, were included as reference strains. All isolates were biochemically identified and characterized by serotyping, phage typing, and amplification of the flaA gene by polymerase chain reaction (PCR). Repetitive intergenic consensus (ERIC) sequence-based PCR was used to generate DNA fingerprints. On the basis of ERIC-PCR fingerprints, Listeria spp. strains were divided into three major clusters matching origin of isolation. ERIC-PCR fingerprints of human and animal isolates were different from those of food isolates. In addition, groups I and II included ten L. monocytogenes strains, and only one Listeria seeligeri strain. Group III included nine L. innocua strains and four L. monocytogenes strains. Computer evaluation of ERIC-PCR fingerprints allowed discrimination between the tested serotypes 1/2b, 4b, 6a, and 6b within each major cluster. The index of discrimination calculated was 0.94. This study suggests that the ERIC-PCR technique provides an alternative method for the identification of Listeria species and the discrimination of strains within one species.

En este estudio se analizaron 24 cepas de Listeria spp. De ellas, 22 fueron obtenidas en San Luis (Argentina), a partir de muestras humanas, de animales y alimentos. Se incluyeron 2 cepas de referencia Listeria monocytogenes CLIP 22762 y Listeria innocua CLIP 74915. Todos los aislamientos fueron identificados bioquímicamente y caracterizados por serotipificación, fagotipificación y detección del gen flaA por reacción en cadena de la polimerasa (PCR). Se generaron perfiles de bandas de ADN mediante la amplificación de secuencias repetitivas de consenso intergénico de enterobacterias (ERIC-PCR). De acuerdo a los resultados obtenidos por ERIC-PCR, las cepas de Listeria spp. fueron divididas en 3 grupos según su origen. Los perfiles de los aislamientos humanos y animales fueron distintos de los correspondientes a alimentos. Por otra parte, dentro de los grupos I y II se incluyeron 10 cepas de L. monocytogenes y solamente una de Listeria seeligeri. Dentro del grupo III no sólo estuvieron incluidas las 9 cepas de L. innocua sino también 4 de L. monocytogenes. La evaluación de los perfiles de bandas obtenidos por ERIC-PCR permitió la discriminación entre los serotipos ensayados 1/2b, 4b, 6a y 6b dentro de cada grupo. El índice de discriminación calculado para ERIC-PCR fue de 0,94. Los resultados sugieren que la técnica de ERIC-PCR provee un método alternativo válido para la identificación de especies de Listeria y, asimismo, permite la diferenciación de cepas dentro de una misma especie.

Deep venous thrombosis: leukocyte rheology at baseline and after in vitro activation.

We evaluated leukocyte rheology, expressed as leukocyte filtration, polymorphonuclear (PMN) membrane fluidity and cytosolic Ca2+ concentration in subjects with acute deep venous leg thrombosis (DVT). In 14 subjects with leg DVT we examined the leukocyte filtration [unfractionated, mononuclear cells (MN), PMNs], PMN membrane fluidity and PMN cytosolic Ca2+ concentration. Subsequently, we evaluated the same PMN variables after in vitro chemotactic activation with 4-phorbol-12-myristate-13-acetate. At baseline, we observed a significant difference in the filtration of unfractionated and MNs and in PMN cytosolic Ca2+ concentration. After PMN activation, a significant variation, greater in DVT subjects, was present in PMN filtration at 5 and 15 min. In normals, no variation was present in PMN membrane fluidity or cytosolic Ca2+ concentration after activation, while in subjects with DVT we found a significant variation in both PMN parameters. These results underline that there is a systemic leukocyte functional alteration in DVT.

Leukocyte rheology before and after chemotactic activation in some venous diseases.

OBJECTIVE: to evaluate leukocyte rheology, polymorphonuclear leukocyte (PMN) membrane fluidity and cytosolic Ca2+ concentration in subjects with post-phlebitic leg syndrome (PPS) and acute deep-venous leg thrombosis (DVT). SUBJECTS: twenty-two subjects with leg PPS and 14 subjects with leg DVT. METHODS: we evaluated the leukocyte filtration (unfractionated, mononuclear cells (MN) and PMN), the PMN membrane fluidity and the PMN cytosolic Ca2+ concentration. Subsequently, we evaluated the same PMN variables after in vitro chemotactic activation with 4-phorbol 12-myristate 13-acetate (PMA) and N -formyl-methionyl-leucyl-phenylalanine (fMLP). RESULTS: at baseline we observed a significant difference in the filtration variables of unfractionated and MN cells and in PMN cytosolic Ca2+ concentration. After activation, in normal subjects and subjects with PPS and DVT, a significant variation in PMN filtration at 5 and 15 minutes was evident. In normal subjects, no variation was present in PMN membrane fluidity or cytosolic Ca2+ concentration after activation. In subjects with PPS and DVT, we found a decrease in PMN membrane fluidity and an increase in PMN cytosolic Ca2+ concentration. After PMN activation (at 5 and 15 min) Delta% of IRFR distinguished normal subjects from subjects with PPS and DVT, while no difference was found in Delta% of membrane fluidity or cytosolic Ca2+ concentration. CONCLUSIONS: there is a functional alteration of leukocytes in these patients whose mechanisms are not yet clear.

Diabetes mellitus: polymorphonuclear leukocyte (PMN) filtration parameters and PMN membrane fluidity after chemotactic activation.

The goal of this research was to determine leukocyte rheology at baseline and after chemotactic activation in type I and type II diabetics. In 19 normal subjects, 21 type I diabetics, and 16 type II diabetics at baseline and after in vitro chemotactic activation (prolonged for 5 and 15 minutes) with two stimulating agents (4-phorbol 12-myristate 13-acetate [PMA] and N-formyl-methionyl-leucyl-phenylalanine [fMLP]), we evaluated polymorphonuclear (PMN) filtration parameters (using a St. George filtrometer [Carri-Med, Dorking, UK] and considering the initial relative flow rate [IRFR] and the concentration of clogging particles [CP]) and PMN membrane fluidity (obtained by marking PMNs with the fluorescent probe 1-(4-[trimethylamino]phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). At baseline, there was a difference between normals and type I and II diabetics for PMN membrane fluidity only. After activation in normals and diabetics of both types, a significant variation was present in PMN filtration parameters (IRFR and CP) at both 5 and 15 minutes. In normals, no variation was present in PMN membrane fluidity after activation with PMA or fMLP. After PMN activation, only in type I diabetics was a significant decrease in PMN membrane fluidity present at both 5 and 15 minutes. After PMN activation with either PMA or fMLP in comparison to basal values, only the mean variation (delta%) of the IRFR was significantly different between normals, type I diabetics, and type II diabetics at both 5 and 15 minutes. From the data obtained, it is evident that after activation, the PMN filtration pattern shows a specific behavior in diabetics of both types, while PMN membrane fluidity changes only in type I diabetics. The latter finding may be the basis of a metabolic pattern present in PMNs of this type, revealed after in vitro activation.

3,4-Dihydro-2H-1-benzopyran-2-carboxylic acids and related compounds as leukotriene antagonists.

Evaluation of a series of 3,4-dihydro-2H-1-benzopyran-2-carboxylic acids linked to the 2-hydroxyacetophenone pharmacophore present in the standard peptidoleukotriene antogonist FPL 55712 (1) has led to the discovery of Ro 23-3544 (7), an antagonist possessing greater potency and duration of action vs LTD4 than the standard (aerosol route of administration, guinea pig bronchoconstriction model). Interestingly, this compound also potently inhibited bronchoconstriction induced by LTB4 whereas 1 did not. Attempts to establish structure--activity relationships in this series involved modifications in the 2-hydroxyacetophenone moiety, the linking chain, and the chroman system. All variations produced analogues which were either inactive or possessed reduced potency relative to acid 7. Optical resolution of 7 was achieved by two methods. Absolute configurations of the enantiomers were determined via X-ray crystallographic analyses of an intermediate as well as a salt of the S enantiomer. Although the enantiomers exhibited similar potencies in in vitro assays and in vivo when administered intravenously, significant differences were observed in the guinea pig bronchoconstriction model vs LTC4 and LTD4 when administered by the aerosol route (S antipode 15-fold more potent). The properties of 7 have been compared with several recently reported leukotriene antagonists.

Analgesic activity of novel spiro heterocycles. 2-Amino-7-oxa-3-thia-1-azaspiro[5,5]undec-1-enes and related compounds.

(+/-)-2-Amino-7-oxa-3-thia-1-azaspiro[5,5]undec-1-ene (1a) and many of its derivatives exhibit significant activity in the phenylquinone writhing and yeast inflamed foot assays. In order to develop structure-activity relationships, the related spiroheterocycles, (+/-)-2-amino-7-oxa-3-thia-1-azaspiro[5,4]dec-1-ene (2), (+/-)-2-amino-3-thia-1-azaspiro[5,5]undec-1-ene (3), and (+/-)-2-amino-7-oxa-1-azaspiro[5,5]undec-1-ene (4), were examined. Of these, only 4 failed to show activity indicating that the analgetic properties displayed by compounds 1--3 are associated, mainly, with the 2-amino-1,3-thiazine ring system. In the 2-acylimino series, evidence is presented suggesting a contribution to the observed activity on the part of the spiroannulated ether ring as well. Both 1a and its p-fluorobenzoyl derivative 33 exhibit analgesic activity in the rat tail-flick assay.