Place of (18)F-FDG-PET with computed tomography in the diagnostic algorithm of patients with fever of unknown origin.

There is evidence for the interest of (18)F-fluoro-deoxyglucose positron emission tomography with computed tomography ((18)F-FDG-PET/CT) in fever of unknown origin (FUO) clinical investigation. However, little and conflicting data exist about its place in the investigation procedure. The aim of this work was to evaluate the clinical value of (18)F-FDG-PET/CT in patients with FUO and identify patients who need early (18)F-FDG-PET/CT rather than a last-resort procedure. We performed a 2-year retrospective cohort study at the Nîmes University Hospital, France. A total of 79 patients (36 men, 43 women, mean age 54.0 ± 16.2 years) with FUO underwent (18)F-FDG-PET/CT. A final diagnosis was established in 61 (77.2 %) cases. Aetiologies of FUO were determined using (18)F-FDG-PET/CT findings in 45 (73.8 % of patients with diagnosis) cases. The sensibility and specificity value were 98 % and 87 %, respectively. The presence of adenopathy, low haemoglobin and increased C-reactive protein (CRP) were predictors of high-yield (18)F-FDG-PET/CT. (18)F-FDG-PET/CT may help to detect most causes of FUO. The predictors of high-yield (18)F-FDG-PET/CT found in this study can help identify patients likely to benefit from specific and early imaging techniques.

Long-term outcome of chronic hepatitis C in a population-based cohort and impact of antiviral therapy: a propensity-adjusted analysis.

This population-based study aimed to assess the determinants of the outcome of chronic hepatitis C with analysis of the impact of antiviral therapy with or without sustained virological response (SVR) on cirrhosis decompensation, hepatocellular carcinoma, liver-related and non-liver-related mortality. A total of 1159 HCV-positive patients newly detected between 1994 and 2001 were included. For each outcome, the prognostic effect of patients' baseline characteristics was estimated by time-dependent Cox models using age as the time-scale and adjusting for treatment received during follow-up. The impact of antiviral therapy was assessed by using a propensity score in a sample including 184 patients treated in the first 24 months following diagnosis who were matched to 184 untreated patients. At the end of a 59-month median follow-up, 100 cases of compensated disease, 58 liver cancer and 163 deaths (55 liver related) were recorded. The 5-year rates of decompensated cirrhosis, hepatocellular carcinoma, liver-related and non-liver-related death were 4.4%, 2.7%, 5.0% and 8.9%, respectively. Multivariate analyses identified two variables with pejorative influence: alcohol consumption (RR = 4.29 for CD; RR = 5.76 for HCC; RR = 6.69 for liver-related death; P < 0.0001); HCV diagnosis unrelated to systematic screening (RR = 2.25 for CD; RR = 3.05 for HCC; RR = 4.31 for liver-related death, P < 0.03). In the matched subset, no significant benefit of antiviral therapy was observed. Nevertheless, among the 144 patients who achieved SVR, no death was observed. This population-based study showed substantial rates of decompensated cirrhosis, hepatocellular carcinoma and non-liver-related mortality. Alcohol consumption and absence of systematic screening were significant determinants of poor outcome, whereas treatment did not have significant influence.

False-positive Aspergillus real-time PCR assay due to a nutritional supplement in a bone marrow transplant recipient with GVH disease.

PCR screening for circulating DNA, especially when combined with antigen testing, has shown promise for the definitive diagnosis of invasive aspergillosis. False positives for Aspergillus real-time PCR assays have been described in several reports, but no sources of fungal DNA contamination could be clearly identified. We report a false-positive case for both galactomannan (GM) antigenemia and Aspergillus PCR due to nutritional supplement intake in a bone marrow transplant recipient with digestive graft-versus-host disease. Our case report also suggests that fungal DNA can pass into the serum from the intestinal tract in the same way as fungal GM. Clinicians should be aware of this possibility, so that the administration of costly, unnecessary antifungal treatments with potential adverse side-effects can be avoided.

Evaluation of a new gridding method for fully 3D direct Fourier PET reconstruction based on a two-plane geometry.

This study investigated how the choice of fixed planes for the representation of the projection data of a cylindrical positron emission tomography (PET) scanner simplifies the frequency interpolation required by the 3D Fourier slice theorem (3D-FST). A new gridding algorithm based on a two-plane geometry and requiring only 1D interpolations in the Fourier domain was compared with the direct implementation of the 3D-FST. We show that the use of two orthogonal planes leads to signal to noise ratios similar to those achieved with the 3D-FST algorithm from projection data acquired with up to two times more count rates, while the resolution remains similar.

Control of the duration of urinary catheterization: impact on catheter-associated urinary tract infection.

Catheter-associated urinary tract infection (CAUTI) is one of the most common types of nosocomial infection. Reducing the duration of catheterization may be a key intervention in CAUTI prevention. We carried out a prospective time-sequence, non-randomized intervention study in five departments of our university hospital. We wished to determine the impact of daily reminders from nurses to physicians to remove unnecessary urinary catheters four days after insertion. The intervention significantly decreased the duration of catheterization in two out of five departments. The frequency of late CAUTI (LCAUTI) among catheterized patients in all five departments decreased from 10.6 to 1.1 per 100 patients (P = 0.003) and the incidence of LCAUTI decreased from 12.3 to 1.8 per 1000 catheter-days (P = 0.03). Logistic regression analysis showed that duration of catheterization and iterative catheter changes were associated with LCAUTI. This study demonstrates that a simple measure can reduce the frequency of LCAUTI.

Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency.

Clinical applications of gene therapy mainly depend on the development of efficient gene transfer vectors. Large DNA molecules can only be transfected into cells by using synthetic vectors such as cationic lipids and polymers. The present investigation was therefore designed to explore the physicochemical properties of cationic lipid-DNA particles, with plasmids ranging from 900 to 52 500 bp. The colloidal stability of the lipoplexes formed by complexing lipopolyamine micelles with plasmid DNA of various lengths, depending on the charge ratio, resulted in the formation of three domains, respectively corresponding to negatively, neutrally and positively charged lipoplexes. Lipoplex morphology and structure were determined by the physicochemical characteristics of the DNA and of the cationic lipid. Thus, the lamellar spacing of the structure was determined by the cationic lipid and its spherical morphology by the DNA. The main result of this study was that the morphological and structural features of the lipopolyamine-DNA complexes did not depend on plasmid DNA length. On the other hand, their gene transfer capacity was affected by the size of plasmid DNA molecules which were sandwiched between the lipid bilayers. The most effective lipopolyamine-DNA complexes for gene transfer were those containing the shortest plasmid DNA.

Biosynthesis of vitamin B12: the multi-enzyme synthesis of precorrin-4 and factor IV.

BACKGROUND: In order to study the biosynthesis of vitamin B12, it is necessary to produce various intermediates along the biosynthetic pathway by enzymic methods. Recently, information on the organisation of the biosynthetic pathway has permitted the selection of the set of enzymes needed to biosynthesise any specific identified intermediate. The aim of the present work was to use recombinant enzymes in reconstituted multi-enzyme systems to biosynthesise particular intermediates. RESULTS: The products of the cobG and cobJ genes from Pseudomonas denitrificans were expressed heterologously in Escherichia coli to afford good levels of activity of the corresponding enzymes, CobG and CobJ. Aerobic incubation of precorrin-3A with the CobG enzyme alone yielded precorrin-3B. When CobJ and S-adenosyl-L-methionine were included in the incubation, the product was precorrin-4. Both precorrin 3B and precorrin-4 are known precursors of vitamin B12 and their availability has allowed new mechanistic studies of enzymic transformations. CONCLUSIONS: Our results show that the expression of the CobG and CobJ enzymes has been successful, thus facilitating the biosynthesis of two precursors of vitamin B12. This lays the foundation for the structure determination of CobG and CobJ as well as future enzymic experiments focusing on later steps of vitamin B12 biosynthesis.

Application of lipids and plasmid design for gene delivery to mammalian cells.

Cationic lipids are widely used for in vitro gene transfer due to their efficiency. The major challenges for the improvement of in vivo cationic lipid-mediated gene delivery reside in the design of more biocompatible lipoplexes mimicking viral-mediated gene delivery and in understanding the fate of the lipoplexes within the cells.

Construction of a broad-host-range non-mobilizable stable vector carrying RP4 par-region.

Plasmid pXL1635 was constructed from the already segregationally stable incP-derived pRK290. Plasmid pXL1635 should be suitable for industrial and environmental uses in Gram- bacteria since (i) it contains the par fragment from RP4 which increases its stability in Pseudomonas denitrificans, a cobalamin-producing and industrially used bacterium, and (ii) the RK2 oriT has been deleted, leading to a non-mobilizable plasmid.

Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.: gene cloning and overexpression, purification and primary structure.

An aliphatic nitrilase, active on adiponitrile and cyanovaleric acid, was identified and purified from Comamonas testosteroni sp. (Ct). Oligodeoxyribonucleotide probes were designed from limited amino acid (aa) sequence information and used to clone the corresponding gene, named nitA. High homologies were found at the aa level between Ct nitrilase and the sequences of known nitrilases. Multi-alignment of sequenced nitrilases suggests that Cys163 of Ct plays an essential role in the active site. This hypothesis is strengthened by molecular studies on nitrilases from Alcaligenes faecalis JM3, and Rhodococcus rhodochrous J1 and K22 [Kobayashi et al., Proc. Natl. Acad. Sci. USA 90 (1993) 247-251; J. Biol. Chem. 267 (1992) 20746-20751; Biochemistry 31 (1992) 9000-9007]. Large amounts of an active recombinant enzyme could be produced in Escherichia coli when nitA was overexpressed together with the E. coli groESL genes.

Cloning and primary structure of the wide-spectrum amidase from Brevibacterium sp. R312: high homology to the amiE product from Pseudomonas aeruginosa.

A Brevibacterium sp. R312 DNA fragment encoding the wide-spectrum amidase (EC 3.5.1.4) has been cloned and sequenced, using limited amino acid (aa) sequence information obtained from the purified enzyme. The deduced aa sequence showed more than 80% strict identity with the Pseudomonas aeruginosa aliphatic amidase, the product of the amiE gene, suggesting a horizontal transfer of the gene during evolution between Gram+ and Gram- bacteria.

Spinal tuberculosis in adults. A study of 103 cases in a developed country, 1980-1994.

Spinal tuberculosis (TB) accounts for about 2% of all cases of TB. New methods of diagnosis such as magnetic resonance imaging (MRI) or percutaneous needle biopsy have emerged. Two distinct patterns of spinal TB can be identified, the classic form, called spondylodiscitis (SPD) in this article, and an increasingly common atypical form characterized by spondylitis without disk involvement (SPwD). We conducted a retrospective study of patients with spinal TB managed in the area of Paris, France, between 1980 and 1994 with the goal of defining the characteristics of spinal TB and comparing SPD to SPwD. The 103 consecutive patients included in our study had TB confirmed by bacteriologic and/or histologic studies of specimens from spinal or paraspinal lesions (93 patients) or from extraspinal skeletal lesions (10 patients). Sixty-eight percent of patients were foreign-born subjects from developing countries. None of our patients was HIV-positive. SPD accounted for 48% of cases and SPwD for 52%. Patients with SPwD were younger and more likely to be foreign-born and to have multiple skeletal TB lesions. Neurologic manifestations were observed in 50% of patients, with no differences between the SPD and SPwD groups. Of the 44 patients investigated by MRI, 6 had normal plain radiographs; MRI was consistently positive and demonstrated epidural involvement in 77% of cases. Bacteriologic and histologic yields were similar for surgical biopsy (n = 16) and for percutaneous needle aspiration and/or biopsy (n = 77). Cultures for Mycobacterium tuberculosis were positive in 83% of patients, and no strains were resistant to rifampin. Median duration of antituberculous chemotherapy was 14 months. Surgical treatment was performed in 24% of patients. There were 2 TB-related deaths. Our data suggest that SPwD may now be the most common pattern of spinal TB in foreign-born subjects in industrialized countries. The reasons for this remain to be elucidated.

Synthesis, activity, and structure--activity relationship studies of novel cationic lipids for DNA transfer.

We have designed and synthesized original cationic lipids for gene delivery. A synthetic method on solid support allowed easy access to unsymmetrically monofunctionalized polyamine building blocks of variable geometries. These polyamine building blocks were introduced into cationic lipids. To optimize the transfection efficiency in the novel series, we have carried out structure-activity relationship studies by introduction of variable-length lipids, of variable-length linkers between lipid and cationic moiety, and of substituted linkers. We introduce the concept of using the linkers within cationic lipids molecules as carriers of side groups harboring various functionalities (side chain entity), as assessed by the introduction of a library composed of cationic entities, additional lipid chains, targeting groups, and finally the molecular probes rhodamine and biotin for cellular traffic studies. The transfection activity of the products was assayed in vitro on Hela carcinoma, on NIH3T3, and on CV1 fibroblasts and in vivo on the Lewis Lung carcinoma model. Products from the series displayed high transfection activities. Results indicated that the introduction of a targeting side chain moiety into the cationic lipid is permitted. A primary physicochemical characterization of the DNA/lipid complexes was demonstrated with this leading compound. Selected products from the series are currently being developed for preclinical studies, and the labeled lipopolyamines can be used to study the intracellular traffic of DNA/cationic lipid complexes.

6-O-alpha-L-Arabinopyranosyl-beta-D-glucopyranosides as aroma precursors from passion fruit.

The 6-O-alpha-L-Arabinopyranosyl-beta-D-glucopyranosides of linalool, benzyl alcohol and 3-methyl-but-2-en-1-ol were isolated from passion fruit (Passiflora edulis) by adsorption chromatography on XAD-2 resin, then further extracted on the same resin after partial enzymic hydrolysis and semi-preparative chromatography on RP-18 phase by HPLC. Their structures were identified by 1H NMR spectroscopy and mass spectral analysis and by methylation analysis of the carbohydrate moieties.

Cluster organization of the genes of Streptomyces pristinaespiralis involved in pristinamycin biosynthesis and resistance elucidated by pulsed-field gel electrophoresis.

Streptomyces pristinaespiralis synthesizes pristinamycin, a member of the streptogramin antibiotic family which consists of a mixture of two types of chemically unrelated compounds named pristinamycins I and pristinamycins II. In order to estimate the size of the Strep. pristinaespiralis chromosome and to elucidate the organization of the pristinamycin biosynthetic and resistance genes already identified, it was decided to use the pulsed-field gel electrophoresis technique. Results indicate that the Strep. pristinaespiralis chromosome is linear and about 7580 kb, as previously shown for several other Streptomyces species. By hybridization, it could be shown that the biosynthetic and resistance genes for pristinamycins I and pristinamycins II, except for the multidrug resistance gene ptr, are interspersed and seem to be organized as a single large cluster, covering less than 200 kb corresponding to 2.6% of the total size of the chromosome. The consequences and significance of such a genetic organization are discussed.

Effect of pH on retention of aroma compounds by beta-lactoglobulin.

Interactions between volatile compounds and BLG in aqueous solution were studied using static and dynamic headspace techniques (exponential dilution). The intensity of interactions between methyl ketones (C7-C9), ethyl esters (C6-C9), limonene, myrcene, and beta-lactoglobulin (BLG) were estimated by determination of the relative infinite dilution activity coefficients (gamma(r)). For a constant pH value, the methyl ketones retention by BLG increased significantly with the hydrophobicity of the volatiles, whereas the retention reached a maximum for ethyl octanoate in the ester series, indicating a possible steric hindrance. For limonene and myrcene an unexpected increase in headspace concentration or "salting out" effect was noticed for acid pH. The variations of the retention according to the pH increase of the medium from pH 3 to pH 11 could be related to structural modifications of the BLG. The retention increase observed between pH 3 and pH 9 resulted from the flexibility modification of the protein, allowing better accessibility to the primary or the secondary hydrophobic sites, whereas the dramatic decrease observed at pH 11 was the consequence of the alkaline denaturation of BLG. Electrostatic interactions occurring at pH 7.5 could also explain the observed retention increase.

Changes of volatile compounds during heating of bacuri pulp.

The formation of volatile compounds from precursors or through chemical rearrangement during heat treatment of bacuri pulp at fruit natural pH were studied using simultaneous distillation/extraction (SDE) technique. An increase of the quantities of oxygenated and hydrocarbon terpenes and, to a lesser degree, aldehydes, was observed after SDE at pH 3, relative to the other extraction methods used, SDE at neutral pH and solid phase extraction (SPE). More particularly, linalool, linalool furanoxides, alpha-terpineol, hotrienol, nerol oxide, nerol, and geraniol were isolated in more important quantities after the first treatment than after the others. These results can be partially explained by the hydrolysis of glycosidically bound compounds previously identified in bacuri. Other pathways such as polyol rearrangements were also involved. The formation of linalool and alpha-terpineol was probably the result of the rearrangement of 2,6-dimethyloct-1-ene-3,7-diol. Moreover, it was assumed that oxidation reactions occurred during SDE at pH 3; more particularly, linalool pyranoxides partially resulted from nonenzymatic oxidation of linalool. When SDE was performed at pH 3, an increase of furfural and 4-methoxy-2,5-dimethyl-3(2H)-furanone was noticed. The modifications of the concentration of aliphatic aldehydes, known as lipid oxidation compounds, and of fatty acid esters were in good agreement with the observed decrease of palmitic and linoleic acid concentrations during this treatment. Moreover, important amounts of 2-acetyl-1-pyrroline were found in the SDE extract recovered at pH 7.

Determination of apparent binding constants for aroma compounds with beta-lactoglobulin by dynamic coupled column liquid chromatography.

Apparent binding constants of aroma compounds limonene, alpha- and beta-ionone, and terpenyl acetate, with beta-lactoglobulin (BLG), were determined, using dynamic coupled column liquid chromatography, for pH values varying from 3 to 11. K(a) values varied from 2.61 to 3.21 x 10(3) M(-1) for limonene, indicating a strong interaction with BLG. Similarly, significant and close apparent binding constants were obtained for alpha- and beta-ionone, 1.7 x 10(2) and 4.5 to 5.4 x 10(2) M(-1), respectively. These data indicated that a similar mechanism is involved for the binding of these two molecules. The weaker values obtained at low pH, for alpha-ionone relative to beta-ionone, can be explained by the existence of steric hindrance. An increase of the apparent binding constant was observed, for all the compounds studied, when the pH was increased from 3 to 9. At this pH, an apparent binding constant was obtained for terpenyl acetate (1.04 x 10(2) M(-1)), whereas this determination was not possible at pH 3 and 6. The apparent binding constant increase was in agreement with the decrease of aroma compound relative activity coefficient in the presence of BLG, previously observed at this pH. It indicated a best accessibility to the same binding site. The binding constants of all the aroma compounds studied decreased at pH 11 as a result of the important release of the BLG structure previously reported.

Osteoporotic fractures at presentation of Cushing's disease: two case reports and a literature review.

Osteoporosis is a common complication of Cushing's disease/syndrome. Fractures can be the presenting manifestation. We report two cases and review 28 others from the literature. Clinical, laboratory, and absorptiometry data are reviewed; the vertebrae and ribs were the most common fracture sites, and osteoporosis reversal after treatment varied with age, gender, and symptom duration. The pathophysiology, which remains controversial, is discussed. A search for Cushing's disease/syndrome should be part of the routine evaluation of osteoporosis in children and adults, particularly men.