Preparation and in vitro/in vivo evaluation of nano-sized crystals for dissolution rate enhancement of ucb-35440-3, a highly dosed poorly water-soluble weak base.

Ucb-35440-3 is a new drug entity under investigation at UCB S.A. Due to its physicochemical characteristics, the drug, a poorly water-soluble weak base, shows poor solubility and dissolution characteristics. In rat, the low oral bioavailability (F < 10%) is largely due to poor absorption. In order to enhance the solubility and dissolution characteristics, formulation of ucb-35440-3 as nanocrystals has been achieved in this study. Nanoparticles were prepared using high pressure homogenization and were characterized in terms of size and morphology. In vitro dissolution characteristics were investigated and compared to the un-milled drug in order to verify the theoretical hypothesis on the benefit of increased surface area. In vivo pharmacokinetic evaluation of ucb-35440-3 nanoparticles was also carried out on rats. Crystalline state evaluation before and following particle size reduction was conducted through polarized light microscopy and PXRD to denote any possible transformation to an amorphous state during the homogenization process. Drug chemical stability was also assessed following homogenization. The dissolution rate increased significantly at pH 3.0, 5.0 and 6.5 for ucb-35440-3 nanoparticles. However, the pharmacokinetic profile obtained yielded lower systemic exposure than the un-milled compound (in fed state), this although being thought to be the consequence of the drug and formulation characteristics.

Immunization with gp96 from Listeria monocytogenes-infected mice is due to N-formylated listerial peptides.

N-Formylated (N-f-met) peptides derived from proteins of the intracellular bacterium Listeria monocytogenes generate a protective, H2-M3-restricted CD8 T cell response in C57BL/6 mice. N-f-met peptide-specific CTL were generated in vitro when mice previously immunized with gp96 isolated from donor mice infected with L. monocytogenes were stimulated with these peptides. No significant peptide-specific CTL activity was observed in mice immunized with gp96 from uninfected animals. Masses corresponding to one N-f-met peptide were found by matrix-assisted laser desorption/ionization-mass spectrometry on gp96 isolated from C57BL/6 mice infected with L. monocytogenes, but not on gp96 from noninfected mice. Therefore, bacterial N-f-met peptides from intracellular bacteria can bind to gp96 in the infected host, and gp96 loaded with these peptides can generate N-f-met-peptide-specific CTL. We assume a unique role of gp96 in Ag processing through the H2-M3 pathway.

Estimation of pharmacokinetic parameters of sodium tungstate after multiple-dose during preclinical studies in beagle dogs.

In this paper, an empirical Bayes methodology was used to determine the pharmacokinetic profile of sodium tungstate in beagle dogs after multiple oral dosing using the P-PHARM computer program. The population estimation algorithm used in P-PHARM is an EM-type procedure. Sodium tungstate was administered orally, three times a day, (i) for 11 days (21 and 42 mg/kg per day) to 18 dogs (nine males and nine females) and (ii) for 13 weeks (15, 30 and 60 mg/kg per day) to 28 dogs (14 males, 14 females). Six other dogs received the compound intravenously (25 and 50 mg/kg). Plasma concentration profiles versus time were compatible with a two-compartment model and first-order kinetics. After oral administration, F (0.61+/-0.086 vs. 0.48+/-0.093), and normalized (to a 7-mg/kg dose of sodium tungstate) AUC (54+/-8.4 vs. 41.2+/-8.5 mg/l x h), C(max) (10.6+/-0.49 vs. 8.5+/-0.57 microg/ml) and C(min) (3.04+/-0.23 vs. 2.04+/-0.22 microg/ml), were higher in male than in female dogs. However, the introduction of the gender in the final model did not contribute statistically to an improvement of the fit of the population pharmacokinetic model. In males, t(1/2) elimination averaged 3.1+/-0.56 vs. 2.6+/-0.18 h in females. The duration of treatment did not modify statistically the pharmacokinetic parameters. After repeated multiple oral administration of 15-60 mg/kg per day of sodium tungstate, tungsten plasma concentrations increased in proportion to dose. No dose-dependent changes in pharmacokinetic parameters occurred.

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

Proteome analysis of rat hepatomas: carcinogen-dependent tumor-associated protein variants.

Proteome analysis led to the identification and characterization of tumor-associated protein variants by two-dimensional electrophoresis and mass spectrometry. We focused on comparing the influence of genotoxic nitroso compounds N-methyl-N-nitrosourea, diethylnitrosamine and N-nitrosomorpholine and the nongenotoxic peroxisome proliferator Nafenopin as tumor-inducing agents on the protein pattern of rat hepatomas. We found several tumor-associated variants that represent members of the aldo-keto reductase superfamily. Their induction and/or inhibition was specifically related to the carcinogen used for tumor induction. The most prominent tumor-associated protein, rat aldose reductase-like protein-1 (rARLP-1) (69% sequence identity to lens aldose reductase) and three additional types of rARLP-1 were detected in nitroso compound-induced rat hepatomas, while rat aldo-keto reductase protein-c (Rak-c), a novel tumor-associated variant (65% sequence identity with 3alpha-hydroxysteroid dehydrogenase) was discovered in N-methyl-N-nitrosourea-induced hepatomas only. 3Alpha-hydroxysteroid dehydrogenase and delta4-3-ketosteroid-5beta-reductase, both liver-specific enzymes, were reduced in amount in all hepatomas investigated, independent of their mode of induction. We conclude, that detoxification enzymes like 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) and delta4-3-ketosteroid-5beta-reductase (5beta-Red) might be replaced in hepatomas by tumor-associated proteins that are often present in the embryonal state, like the rARLPs or the Rak-c protein. Their induction appears to reflect an altered constitutive pattern of detoxification enzymes, detoxifying toxic aldehydes being induced by nitroso compounds. In contrast, members of the aldo-keto reductase superfamily have not been found in Nafenopin-induced hepatomas. The pattern of tumor-associated protein variants is apparently characteristic for a given group of initiating carcinogens. The hypothesis is proposed that carcinogens leave specific fingerprints at the proteome level of manifest liver tumors.

Matrix-assisted laser desorption-ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures.

In theory, peptide mass fingerprinting by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower microl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.

Criteria predictive of limb viability at 1 year in patients with chronic severe ischemia--TcPO2 and demographic parameters.

Following an earlier study, the investigators sought to identify and define objective prognostic criteria of viability at 1 year of a limb with severe chronic ischemia. A study was undertaken in 116 patients (118 limbs) (74 men and 42 women), with a mean age of 71.9 years for men and 81.6 years for women. Static transcutaneous oxygen pressure (TcPO2) was measured with a verticalization sensitization test and inhalation of oxygen on JO and viability of the limb noted 1 year later. Logistic analysis was made of 13 oximetry parameters and two demographic parameters (age and gender). Results were analyzed in absolute terms and by tissue oxygenation ratio (TOR) (ratio between absolute TcPO2 at the foot and at a chest reference electrode). Six factors appeared to be prognostic factors of limb viability at 1 year, statistically significant at 6% according to threshold values: age, verticalization TcPO2, TcPO2 after 1 minute's inhalation of oxygen, TcPO2 after 4 minutes' inhalation of oxygen, and slope of TcPO2 and slope of TOR between 1 and 4 minutes' inhalation. A 1 year viability index integrating these criteria is suggested.

Pharmacokinetics of sodium tungstate in rat and dog: a population approach.

Sodium tungstate has been found to correct hyperglycemia in insulin- and noninsulin-dependent models of diabetes when administered in drinking fluid with a low degree of toxicity; thus, it provides a potential treatment for diabetes. In the present report, pharmacokinetic studies with sodium tungstate were carried out in the Sprague-Dawley rat and beagle dog. This drug was administered either i.v. (8.97 mg/kg in rat; 25 and 50 mg/kg in dog) or orally in the form of solution (35.9 and 107.7 mg/kg in rat; 25 and 50 mg/kg in dog). Tungsten was quantified using an inductively coupled plasma method. Pharmacokinetic parameters were estimated using a population approach. Sodium tungstate followed first order kinetics, and plasma concentration-versus-time data were adequately described by a two-compartment model. In rat, bioavailability was high (92%), whereas it was lower in dog (approximately 65%). The total volume of distribution expressed by unit of body weight was much higher when the animal was smaller (0.46 l/kg in rat versus 0.23 l/kg in dog). The total body clearance normalized by weight, 0.19 l/h/kg in rat versus 0.043 l/h/kg in dog, changed as for the volume of distribution. The elimination half-life was two times higher in dog (approximately 4 h) than in rat (approximately 1.7 h). In the range of 35.9 to 107.7 mg/kg after oral administration in rat and 25 to 50 mg/kg after oral and i.v. administration in dog, tungsten plasma concentrations increased in proportion to dose.

Comparative proteome analysis of Helicobacter pylori.

Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2-DE patterns with their identified protein species were added to the dynamic 2D-PAGE database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.

Foraminal epidural extra osseous cavernous hemangioma of the cervical spine: a case report.

STUDY DESIGN: A case report of purely epidural foraminal cervical cavernous angioma assessed by magnetic resonance imaging and diagnosed at pathologic study. OBJECTIVE: To illustrate a rare cause of cervical foraminal mass mimicking a schwannoma. SUMMARY OF BACKGROUND DATA: Several cases of epidural cavernous angioma have been reported. A purely epidural cavernous angioma in a cervical foramen has never been reported in the literature. METHOD: A 36-year-old man sought treatment for acute weakness in his right upper limb with radicular distribution. On magnetic resonance images, the lesion appeared as a foraminal mass with no bone invasion or hematic components. It enhanced after intravenous administration of Gadolinium. Pathologic study after surgery showed a typical cavernous angioma. RESULTS: The patient improved slightly after surgery. CONCLUSION: Clinical and radiologic presentation could be confusing in a purely foraminal epidural cervical cavernous hemangioma. Cavernous hemangioma must be known as a differential diagnosis of a foraminal schwannoma. Diagnosis of cavernous angioma is made easily on pathologic examination.

MRI and ultrasound in children with juvenile chronic arthritis.

In this era of advancing imaging technology, a knowledge of the relative values of available imaging techniques is necessary to optimize the management of children with juvenile chronic arthritis (JCA). After clinical examination, plain films remain the initial investigation. The need for radiation protection must be a priority in children with JCA. Conventional radiographs allow grouping of the various arthritides (on the base of the distribution and pattern of joint space changes) and staging of disease progression. Ultrasound (US) is very sensitive in the detection of joint effusions, especially in the hip, and guides fluid aspiration. US and Doppler can be used for the evaluation of synovial hypertrophy and activity. Arthrography and to a certain extent nuclear studies have been replaced by magnetic resonance imaging (MRI). MRI can demonstrate articular cartilage, joint effusion, synovial hypertrophy, cortical and medullary bone, cartilage and bone perfusion, and fibrocartilaginous structures (menisci and ligaments). Contrast enhanced MRI is the most sensitive modality to determine whether an arthritic condition is present. However, it does not assist in establishing a specific diagnosis. MRI determines accurately the activity and the extent of the disease and is particularly useful in the early detection of articular damage. Finally, MRI is of major importance in the evaluation of response to local therapy (especially steroids) and the detection of complications.

Analysis of missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation after in-gel tryptic digestion.

Peptide mass fingerprinting is a powerful tool for the identification of proteins. Trypsin is the most widely used enzyme for this purpose. Therefore, 104 protein digests from human Jurkat T cells and Mycobacterium were analyzed considering missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation. About 90% of the matched peptides with missed cleavage sites could be classified into three groups: (i) lysine and arginine with a neighbouring proline on the carboxy-terminal side, (ii) neighboring lysines/arginines, and (iii) lysines and arginines with an aspartic acid or glutamic acid residue on either the amino- or carboxy-terminal side. The first group is already accounted for by search programs. The number of missed cleavage sites can be increased without reducing the precision of the database search by taking the other two groups into consideration. Peptides with tryptophan were observed in non, singly (+16 Da) and doubly (+32 Da) oxidized forms. The higher oxidized form was only observed with lower intensity in the presence of the lower oxidized form. Peptides with N-terminal glutamine were found always as pyroglutamate (-17 Da), and in the majority of cases in pairs with unmodified glutamine. These data can be used for the refinement of protein searches by peptide mass fingerprinting.

Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens.

In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non-virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two-dimensional electrophoresis (2-DE) combining non-equilibrium pH gradient electrophoresis (NEPHGE) with SDS-PAGE. Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2-DE database (http://www.mpiib-berlin.mpg.de/2D-PAGE). Silver-stained 2-DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identified and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.

A dynamic two-dimensional polyacrylamide gel electrophoresis database: the mycobacterial proteome via Internet.

Proteome analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry, in combination with protein chemical methods, is a powerful approach for the analysis of the protein composition of complex biological samples. Data organization is imperative for efficient handling of the vast amount of information generated. Thus we have constructed a 2-D PAGE database to store and compare protein patterns of cell-associated and culture-supernatant proteins of different mycobacterial strains. In accordance with the guidelines for federated 2-DE databases, we developed a program that generates a dynamic 2-D PAGE database for the World-Wide-Web to organise and publish, via the internet, our results from proteome analysis of different Mycobacterium tuberculosis as well as Mycobacterium bovis BCG strains. The uniform resource locator for the database is http://www.mpiib-berlin.mpg.de/2D-PAGE and can be read with a Java compatible browser. The interactive hypertext markup language documents displayed are generated dynamically in each individual session from a rational data file, a 2-D gel image file and a map file describing the protein spots as polygons. The program consists of common gateway interface scripts written in PERL, minimizing the administrative workload of the database. Furthermore, the database facilitates not only interactive use, but also worldwide active participation of other scientific groups with their own data, requiring only minimal computer hardware and knowledge of information technology.

Cystography after the Cohen ureterovesical reimplantation: is it necessary at a training center?

PURPOSE: Reimplantation by the Cohen procedure has a low rate of recurrent reflux, although postoperative cystography is done routinely at most centers. According to the French training program for pediatric surgery and urology residents, reimplantation is the main pediatric urology procedure performed during residency. We determine whether it is necessary to perform postoperative cystography routinely and whether the fact that the procedure is done by a junior surgeon modifies management. MATERIALS AND METHODS: A total of 268 children with primary vesicoureteral reflux underwent ureteral reimplantation by the Cohen transtrigonal technique. Bilateral reimplantation was done in 97% of the cases. Reimplantation was performed by a surgery resident assisted by a clinical fellow or senior consultant surgeon in 37% of the cases. Routine cystography and renal ultrasound were done in all patients postoperatively. Followup ranged from 6 months to 5 years (mean 10 months). RESULTS: In 2 children (0.7%) with recurrent reflux surgery was not performed by a resident. One of the 2 children had asymptomatic persistent reflux and no further surgery was done. In the other child postoperative cystography was normal at 6 months. One year later she had acute pyelonephritis with recurrent unilateral reflux and underwent repeat reimplantation. CONCLUSIONS: Routine cystography is not necessary after bilateral Cohen reimplantation. Reflux recurrence is low even at a training center where surgery may be performed by junior surgeons.

[Radio-anatomy of lymph node groups in the area of otolaryngology]. / Radio-anatomie des chaînes ganglionnaires de la sphère ORL.

Imaging plays an essential role in the management of head and neck lymph node. Knowledge of the anatomy is indispensible to interpret ultrasound, CT, and MR examination.

Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis patterns by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS).

Identification of proteins separated by two-dimensional electrophoresis (2-DE) is a necessary task to overcome the purely descriptive character of 2-DE and a prerequisite to the construction of 2-DE databases in proteome projects. Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has a sensitivity for peptide detection in the lower fmol range, which should be sufficient for an analysis of even weakly silver-stained protein spots by peptide mass fingerprinting. Unfortunately, proteins are modified by the silver staining procedure, leading to low sequence coverage. Omission of glutaraldehyde increased the sequence coverage, but this improved sequence coverage is still clearly below the sequence coverage starting with Coomassie Brilliant Blue (CBB) R-250-stained spots. Other factors additionally seem to modify proteins during silver staining. By decreasing the protein amount, the advantage of very sensitive detection on the gel is lost during identification, because the resulting low sequence coverage is not sufficient for secure identification. Low-quantity proteins can be identified better starting with CBB G-250 or Zn-imidazol-stained proteins. In contrast, for high-quantity CBB R-250-stained spots, a sequence coverage of up to 90% can be obtained by using only one cleaving enzyme, and up to 80% was reached for medium-quantity spots after combination of tryptic digest with Asp-N- and Glu-C digest.