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Biomedical subjects

A Pascual

Publications and source records attributed to A Pascual.

At least 73 records · Page 4Linked to original sources
PubMed1999

Increasing mixed haematopoietic chimaerism after BMT with total depletion of CD4+ and partial depletion of CD8+ lymphocytes is associated with a higher incidence of relapse.

In this study we analysed the incidence and clinical impact of the persistence of host haemopoiesis (mixed chimaerism, MC) after allogeneic BMT in 35 consecutive patients with haematologic malignancies using a total CD4+ cell-depleted graft with an adjusted dose of CD8+ cells (1x10(8)/kg). Chimaerism was assessed by PCR amplification of VNTRs in 30 evaluable patients: 19 non-CML and 11 CML cases which were also evaluated for the BCR-ABL transcript by RT-PCR. All but one had complete engraftment with a donor profile early post-BMT. At the end of the study period, 12 of 30 patients displayed MC (40%). The overall disease-free survival for MC patients was clearly unfavourable when compared to those who exhibited a donor profile (24.7% vs. 100%, P = 0.005). However, we found that only two of five patients with MC in the non-CML group relapsed, whereas a clear correlation could be made between MC and relapse in CML (seven showed MC, preceding cytogenetic or haematological relapse in six of them, which displayed a prior BCR-ABL mRNA positivity). In addition, a quantitative-PCR approach enabled us to demonstrate that increasing amounts of MC are invariably associated with subsequent relapse, whereas a low stable level of host or complete donor haemopoiesis is consistent with clinical complete remission. Although these results suggest that the clinical impact of MC may depend on the underlying disease, it is compatible with the concept that the graft-versus-leukaemia effect against CML is mainly exerted by donor CD4+ lymphocytes. Elimination of this cellular subset may be responsible for the inability of the graft to prevent a progressive increase in the tumor cell burden.

Adolescent↗
PubMed1999

In-vitro activity of levofloxacin, ofloxacin and D-ofloxacin against coryneform bacteria and Listeria monocytogenes.

The objective of this study was to evaluate the in-vitro activity of levofloxacin, ofloxacin and D-ofloxacin compared with ciprofloxacin, norfloxacin and sparfloxacin against coryneform bacteria and Listeria monocytogenes isolated from clinical samples. The following organisms (and number of strains) were studied: Corynebacterium jeikeium (20), Corynebacterium urealyticum (20), Corynebacterium minutissimum (20), Corynebacterium striatum (20), Corynebacterium amycolatum (30), Brevibacterium spp. (15) and Listeria monocytogenes (15). Antimicrobial activity was determined by microdilution using cation-adjusted Mueller-Hinton broth supplemented with 0.5% Tween 80 when testing C. jeikeium or C. urealyticum. Fluoroquinolones were used in the range 0.015-16 mg/L. Plates were incubated in air at 35 degrees C for 18-20 h (24 h when testing C. jeikeium or C. urealyticum). The following MIC50 values were obtained for all 140 organisms tested: levofloxacin, 1 mg/L; ofloxacin, 2 mg/L; D-ofloxacin, > 16 mg/L; ciprofloxacin, 1 mg/L; norfloxacin, 16 mg/L; sparfloxacin, 1 mg/L. MIC90 values were > 16 mg/L for all test antibiotics with the exception of levofloxacin, which had an MIC90 value of 16 mg/L. At a concentration of 2 mg/L, levofloxacin inhibited all L. monocytogenes strains and 35-93% of the remaining species. MIC90 values of ofloxacin were one dilution step higher than those of levofloxacin against C. minutissimum, C. striatum, Brevibacterium spp. and L. monocytogenes. Levofloxacin showed similar (C. jeikeium, C. urealyticum, C. amycolatum and Brevibacterium spp.) or greater (C. minutissimum and C. striatum) activity than ciprofloxacin and sparfloxacin, and higher than D-ofloxacin or norfloxacin against all species studied. In conclusion, levofloxacin was the most active of the six fluoroquinolones evaluated against coryneform bacteria isolated from clinical samples and could therefore be a promising treatment option in this setting.

Anti-Infective Agents↗
PubMed1999

Uptake and intracellular activity of moxifloxacin in human neutrophils and tissue-cultured epithelial cells.

The penetration by moxifloxacin of human neutrophils (polymorphonuclear leukocytes [PMN]) and tissue-cultured epithelial cells (McCoy cells) was evaluated by a fluorometric assay. At extracellular concentrations of 5 mg/liter, the cellular-to-extracellular concentration ratios (C/E) of moxifloxacin in PMN and McCoy cells were 10.9 +/- 1.0 and 8.7 +/- 1.0, respectively (20 min; 37 degrees C). The uptake of moxifloxacin by PMN was rapid, reversible, nonsaturable (at extracellular concentrations ranging from 1 to 50 microg/ml), and not affected by cell viability. The uptake of moxifloxacin was affected by external pH and the environmental temperature. The incubation of PMN in the presence of sodium fluoride, sodium cyanide, and carbonyl cyanide m-chlorophenylhydrazone significantly decreased the C/E of this agent. Neither PMN stimulation nor phagocytosis of opsonized Staphylococcus aureus significantly affected the uptake of moxifloxacin by human PMN. This agent, at concentrations of 0.5, 1, and 5 mg/liter, induced a significant reduction in the survival of intracellular S. aureus in human PMN. In summary, moxifloxacin reaches much higher intracellular concentrations within phagocytic and nonphagocytic cells than extracellular ones, remaining active inside the neutrophils.

Anti-Infective Agents↗
PubMed1999

Resistance of Pseudomonas aeruginosa to imipenem induced by eluates from siliconized latex urinary catheters is related to outer membrane protein alterations.

The activity of imipenem against Pseudomonas aeruginosa HUS-3 decreased by 16 times in the presence of substances eluted from siliconized latex urinary catheters (SLUCs). SLUCs did not inactivate imipenem or increase beta-lactamase activity. The outer membrane of P. aeruginosa HUS-3 grown in the presence of eluate lacked an OprD-like protein and expressed a new 50-kDa protein. The decreased activity of imipenem against P. aeruginosa in the presence of SLUCs is related to the loss of an OprD-like protein and the expression of a new outer membrane protein.

Drug Resistance, Microbial↗
PubMed1999

Development of resistance during antimicrobial therapy caused by insertion sequence interruption of porin genes.

We have demonstrated by using an in vitro approach that interruption of the OmpK36 porin gene by insertion sequences (ISs) is a common type of mutation that causes loss of porin expression and increased resistance to cefoxitin in Klebsiella pneumoniae. This mechanism also operates in vivo: of 13 porin-deficient cefoxitin-resistant clinical isolates of K. pneumoniae, 4 presented ISs in their ompK36 gene.

Bacterial Proteins↗
PubMed1999

Roles of beta-lactamases and porins in activities of carbapenems and cephalosporins against Klebsiella pneumoniae.

Two clinical isolates of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae were noted to be less susceptible than expected to imipenem. Both were missing outer membrane proteins that serve as channels for antibiotic entry. The role of beta-lactamase in resistance was investigated by eliminating the original ESBL and introducing plasmids encoding various ESBLs and AmpC beta-lactamase types, by studying the effect of an increased inoculum, and by evaluating interactions with beta-lactamase inhibitors. The contribution of porin deficiency was investigated by restoring a functional ompK36 gene on a plasmid. Plasmids encoding AmpC-type beta-lactamases provided resistance to imipenem (up to 64 microg/ml) and meropenem (up to 16 microg/ml) in strains deficient in porins. Carbapenem resistance showed little inoculum effect, was not affected by clavulanate but was blocked by BRL 42715, and was diminished if OmpK36 porin was restored. Plasmids encoding TEM- and SHV-type ESBLs conferred resistance to cefepime and cefpirome, as well as to earlier oxyimino-beta-lactams. This resistance was magnified with an increased inoculum, was blocked by clavulanate, and was also lowered by OmpK36 porin restoration. In addition, SHV-2 beta-lactamase had a small effect on carbapenem resistance (imipenem MIC, 4 microg/ml, increasing to 16 microg/ml with a higher inoculum) when porins were absent. In K. pneumoniae porin loss can thus augment resistance provided either by TEM- or SHV-type ESBLs or by plasmid-mediated AmpC enzymes to include the latest oxyimino-beta-lactams and carbapenems.

Carbapenems↗
PubMed1999

Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme.

The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.

Amino Acid Sequence↗
PubMed1998

Thyroid hormone negatively regulates the transcriptional activity of the beta-amyloid precursor protein gene.

The expression of the beta-amyloid precursor protein (APP), which plays a key role in the development of Alzheimer's disease, is regulated by a variety of cellular mediators in a cell-dependent manner. In the present study, we present evidence that thyroid hormones negatively regulate the expression of the APP gene in neuroblastoma cells. Transient transfection studies using plasmids that contain progressive deletions of the 5' region of the gene demonstrate that triiodothyronine (T3), the more active form of the thyroid hormones, represses APP promoter activity by a mechanism that requires binding of the nuclear T3 receptor (TR) to a specific sequence located in the first exon. The unliganded receptor increases promoter activity, and T3 reverses that activity to basal levels. The repressive effect of T3 does not exhibit TR isoform specificity, and it is equally mediated by TRalpha and TRbeta. Gel mobility shift assays using in vitro synthesized nuclear receptors and nuclear extracts led to the identification of a negative thyroid hormone response element, at nucleotide position +80/+96, that preferentially binds heterodimers of TR with the retinoid X receptor. Insertion of sequences containing this element confers negative regulation by T3 to a heterologous TK promoter, thus indicating the functionality of the element.

Amyloid beta-Protein Precursor↗
PubMed1998

Quinolone resistance from a transferable plasmid.

BACKGROUND: Bacteria can mutate to acquire quinolone resistance by target alterations or diminished drug accumulation. Plasmid-mediated resistance to quinolones in clinical isolates has been claimed but not confirmed. We investigated whether a multiresistance plasmid could transfer resistance to quinolones between bacteria. METHODS: We transferred resistance between strains by conjugation. The resistance plasmid was visualised in different hosts by agarose-gel electrophoresis. We determined the frequency of spontaneous mutations to ciprofloxacin or nalidixic-acid resistance in Escherichia coli strains, with or without the quinolone resistance plasmid. FINDINGS: A multiresistance plasmid (pMG252) from a clinical isolate of Klebsiella pneumoniae was found to increase quinolone resistance to minimum inhibitory concentrations (MICs) as high as 32 microg/mL for ciprofloxacin when transferred to strains of K pneumoniae deficient in outer-membrane porins. Much lower resistance was seen when pMG252 was introduced into K pneumoniae or E coli strains with normal porins. The plasmid had a wide host range and expressed quinolone resistance in other enterobacteriaceae and in Pseudomonas aeruginosa. From a plasmid-containing E coli strain with ciprofloxacin MIC of 0.25 microg/mL and nalidixic-acid MIC of 32 microg/mL, quinolone-resistant mutants could be obtained at more than 100 times the frequency of a plasmid-free strain, reaching MICs for ciprofloxacin of 4 microg/mL and for nalidixic acid of 256 microg/mL. INTERPRETATION: Transferable resistance to fluoroquinines and nalidixic acid has been found in a clinical isolate of K pneumoniae on a broad host range plasmid. Although resistance was low in wild-type strains, higher levels of quinolone resistance arose readily by mutation. Such a plasmid can speed the development and spread of resistance to these valuable antimicrobial agents.

Anti-Infective Agents↗
PubMed1998

Pneumocephalus after shunting for hydrocephalus.

A case of tension pneumocephalus that occurred after ventriculoperitoneal shunting is presented. We have reviewed 12 cases of pneumocephalus in association with ventriculoperitoneal shunt placement. This phenomenon occurs when air is forced through the shunt or enters through the cranial base because of: iatrogenic postsurgical connection, congenital fistula, trauma, or thinning of the cranial base. Ways of preventing and treating this problem are outlined.

Child↗
PubMed1998

In vitro activities of ketolide HMR 3647, macrolides, and clindamycin against Coryneform bacteria.

The in vitro activity of ketolide HMR 3647 against coryneform bacteria isolated from clinical samples was evaluated. Except against Corynebacterium jeikeium and C. urealyticum, HMR 3647 showed high activity against Corynebacterium spp., being more active than 14- and 16-membered macrolides, azithromycin, or clindamycin. HMR 3647 also had high in vitro activity against Brevibacterium spp. and Listeria monocytogenes.

Anti-Bacterial Agents↗
PubMed1998

Energy-dependent accumulation of fluoroquinolones in quinolone-resistant Klebsiella pneumoniae strains.

The intracellular accumulation of norfloxacin and pefloxacin in Klebsiella pneumoniae was evaluated. The roles of lipopolysaccharide, capsule, and outer membrane proteins were not important for the intrabacterial accumulation of fluoroquinolones in isogenic strains with known outer membrane alterations. In fluoroquinolone-resistant clinical isolates also expressing GyrA alterations, an active efflux leading to decreased accumulation of the drugs enhanced their resistance to these agents.

Anti-Infective Agents↗
PubMed1998

Thyroid hormones regulate beta-amyloid gene splicing and protein secretion in neuroblastoma cells.

The beta-amyloid protein (Abeta), the major component of the senile plaques found in Alzheimer brains, derives from a larger beta-amyloid precursor protein (APP). Alternative splicing of the APP gene yields three major APP messenger RNAs (mRNAs), which, in turn, give rise to the APP770, APP751, and APP695 protein isoforms. In this study we examined the effects of thyroid hormone on APP expression in N2a-beta neuroblastoma cells. T3 caused a significant increase in the APP770 mRNA band, in detriment of the APP695 mRNA, which was proportionately reduced. In agreement with these results, T3 markedly altered the relative ratio of intracellular APP isoforms, increasing the amount of APP770 and causing an equivalent reduction of the immature APP695 isoform. In accordance with these results, the soluble APP695-derived form was specifically reduced in the culture medium obtained from T3-treated cells. In contrast, the increase in intracellular APP770 was not followed by an enhanced release of soluble derivatives of this isoform. These results suggest that T3 regulates not only APP gene splicing, but also the processing and secretion of the APP peptides. According to our results, thyroid hormone might play a role in the development of Alzheimer's disease by modulating the intracellular and extracellular contents of APP isoforms.

Alternative Splicing↗
PubMed1998

[Laboratory control in diagnosis and treatment of infective endocarditis].

Infectious endocarditis is caused by a wide variety of microorganisms. Etiological diagnosis depends either on direct methods, directed to the detection and isolation of the causative organisms, or serological methods. Blood cultures remain as the best method for the diagnosis of bacterial endocarditis. Blood culture technology has changed due to the appearance of new automatic continuous monitoring systems which shorten the detection time. Endocarditis with a negative blood culture has decreased below 5% because of new blood culture systems, new serological methods and molecular technology. The control of antimicrobial therapy in endocarditis must be performed by expert laboratories. A minimal bactericidal concentration, serum levels of antimicrobials and the serum bactericidal test are the most useful parameters. Their execution requires the use of strict protocols and interpretation must be carried out with an expert in clinical microbiology and infectious diseases.

Anti-Bacterial Agents↗
PubMed1998

[Opsonin requirements for phagocytosis of Enterococcus spp. by human polymorphonuclear leukocytes].

BACKGROUND: Interaction of Enterococcus spp. and host defense mechanisms is not well known. Opsonic requirements of Enterococcus faecalis and Enterococcus faecium to be phagocyted by human polymorphonuclear leukocytes (PMN) were evaluated. METHODS: Twenty strains (10 E. faecalis and 10 E. faecium) were studied. Phagocytosis was determined by a radiometric assay. Bacterial cells were labelled with 3H-adenine and opsonized with: a) 10% of human pool sera (HPS); b) 10% of decomplemented HPS, and c) albumin and fibronectin. RESULTS: Phagocytosis of Enterococcus spp. by PMN in the presence of HPS was significantly higher than that in the absence of opsonins. The phagocytosis of E. faecium was higher than that of E. faecalis. A strain-dependent effect of complement in the phagocytosis of Enterococcus spp. was observed. Neither albumin nor fibronectin showed an opsonic activity on Enterococcus spp. CONCLUSIONS: A great heterogeneity in the opsonic requirements of Enterococcus spp. was observed. Serum opsonins show a critical role in the phagocytosis of E. faecalis and E. faecium by PMN, this effect being more relevant with E. faecium. A strain-dependent opsonic activity of complement was observed.

Adult↗