Selective HSP90ß inhibition results in TNF and TRAIL mediated HIF1α degradation.

Signaling via TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Previous reports demonstrated that pro-survival signaling emanates from membrane resident TNF-R1 complexes (complex I) while only internalized TNF-R1 complexes are capable for DISC formation (complex II) and thus, apoptosis induction. Internalized TNF-R1 containing endosomes undergo intracellular maturation towards lysosomes, resulting in activation and release of Cathepsin D (CtsD) into the cytoplasm. We recently revealed HSP90 as target for proteolytic cleavage by CtsD, resulting in cell death amplification. In this study, we show that extrinsic cell death activation via TNF or TRAIL results in HSP90ß degradation. Co-incubation of cells with either TNF or TRAIL in combination with the HSP90ß inhibitor KUNB105 but not HSP90α selective inhibition promotes apoptosis induction. In an attempt to reveal further downstream targets of combined TNF-R1 or TRAIL-R1/-R2 activation with HSP90ß inhibition, we identify HIF1α and validate its ligand:inhibitor triggered degradation. Together, these findings suggest that selective inhibition of HSP90 isoforms together with death ligand stimulation may provide novel strategies for therapy of inflammatory diseases or cancer, in future.

Identification of a tumor-specific allo-HLA-restricted γδTCR.

γδT cells are key players in cancer immune surveillance because of their ability to recognize malignant transformed cells, which makes them promising therapeutic tools in the treatment of cancer. However, the biological mechanisms of how γδT-cell receptors (TCRs) interact with their ligands are poorly understood. Within this context, we describe the novel allo-HLA-restricted and CD8α-dependent Vγ5Vδ1TCR. In contrast to the previous assumption of the general allo-HLA reactivity of a minor fraction of γδTCRs, we show that classic anti-HLA-directed, γδTCR-mediated reactivity can selectively act on hematological and solid tumor cells, while not harming healthy tissues in vitro and in vivo. We identified the molecular interface with proximity to the peptide-binding groove of HLA-A*24:02 as the essential determinant for recognition and describe the critical role of CD8 as a coreceptor. We conclude that alloreactive γδT-cell repertoires provide therapeutic opportunities, either within the context of haplotransplantation or as individual γδTCRs for genetic engineering of tumor-reactive T cells.

[Ponseti method for treatment of idiopathic clubfoot]. / Klumpfußtherapie nach Ponseti.

OBJECTIVE: Pain-free, plantigrade, functional foot through gentle manipulation without extended surgery and with decreased probability of relapse. INDICATIONS: Idiopathic clubfoot; neurogenic and secondary clubfeet. CONTRAINDICATIONS: None. SURGICAL TECHNIQUE: Simultaneous correction of all components of the clubfoot. Mainly conservative, with serial casts. Slight supination to address the cavus and increasing abduction to align the midfoot bones while putting counter-pressure on the head of the talus. Surgery primarily only to correct the equinus, which can often not be accomplished through casting, and consists of a simple subcutaneous section. Due to tendency to relapse, further surgery might be necessary, followed by serial casting. Remaining deformity can be treated by percutaneous lengthening of the Achilles tendon, percutaneous release of the plantar fascia or a transfer of the tibialis anterior tendon to the third cuneiform. POSTOPERATIVE MANAGEMENT: Abduction orthosis for stabilization of the clinical result 24 h/day for 3 months, then only at night- and naptime through end of the third year of life. Follow-up every 3-4 months.

Serum GFAP is a diagnostic marker for glioblastoma multiforme.

A serum marker for malignant cerebral astrocytomas could improve both differential diagnosis and clinical management of brain tumour patients. To evaluate whether the serum concentration of glial fibrillary acidic protein (GFAP) may indicate glioblastoma multiforme (GBM) in patients with single supratentorial space-occupying lesions, we prospectively examined 50 consecutive patients with histologically proven GBM, World Health Organization (WHO) grade IV, 14 patients with anaplastic astrocytoma (WHO grade III), 4 patients with anaplastic oligodendroglioma, 13 patients with diffuse astrocytoma (WHO grade II), 17 patients with a single cerebral metastasis and 50 healthy controls. Serum was taken from the patients before tumour resection or stereotactic biopsy. Serum GFAP levels were determined using a commercially available ELISA test and were detectable in 40 out of the 50 GBM patients (median: 0.18 microg/l; range: 0-5.6 microg/l). The levels were significantly elevated compared with those of the non-GBM tumour patients and healthy controls (median: 0 mug/l; range: 0-0.024 microg/l; P < 0.0001, respectively). Non-GBM tumour patients and all healthy subjects showed zero serum GFAP levels. There was a significant correlation between tumour volume (Spearman Rho, CC = 0.47; 95% confidence interval, 0.2-0.67; P < 0.001), tumour necrosis volume (CC = 0.49; 95% confidence interval, 0.2-0.72; P = 0.004), the amount of necrotic GFAP positive cells (CC = 0.61; 95% confidence interval, 0.29-0.81; P = 0.007) and serum GFAP level among the GBM patients. A serum GFAP level of >0.05 microg/l was 76% sensitive and 100% specific for the diagnosis of GBM in patients with a single supratentorial mass lesion in this series. Therefore, it can be concluded that serum GFAP constitutes a diagnostic biomarker for GBM. Future studies should investigate whether serum GFAP could also be used to monitor therapeutic effects and whether it may have a prognostic value.

Isolated renal metastasis after colon cancer.

Renal infiltration of colon adenocarcinoma is a rare event. The authors present the case report of a 52-year-old female who had a high carcinoembryonic antigen level 18 months after right hemicolectomy and a chemotherapy regimen to treat transverse colon adenocarcinoma. The patient presented cancer recurrence after 12 months, and underwent a paraaortic lymphadenoctomy and a second adjuvant chemotherapy with the folfox regimen. Abdomen computerized tomography revealed two solid masses in the right kidney, without evidence of any other metastatic sites. A nephrectomy was performed in the right kidney followed by adjuvant chemotherapy.

Quantitative analysis of the novel anticancer drug ABT-518, a matrix metalloproteinase inhibitor, plus the screening of six metabolites in human plasma using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry.

A liquid chromatographic/tandem mass spectrometric (LC/MS/MS) assay for the quantitative analysis of the novel anticancer drug ABT-518 and the screening of six potential metabolites in human plasma has been developed and validated to support a phase I study with the drug. ABT-518 is an inhibitor of matrix metalloproteinases, which are associated with tumor growth and development of metastasis. Plasma samples were prepared for analysis using a simple solid-phase extraction method on phenyl cartridges. LC separation was performed on a Zorbax extend C18 column (150 x 2.1 mm i.d., 5 microm particle size) using a mobile phase of methanol-aqueous 10 mM ammonium hydroxide (80:20, v/v) pumped at a flow-rate of 0.2 ml min(-1). An API2000 triple-quadrupole mass spectrometer was used for specific and sensitive detection. The best chromatographic speed (total run time 8 min) and peak shapes were obtained by employing an alkaline mobile phase (pH in aqueous phase approximately 10). Furthermore, an alkaline eluent was favored in order to obtain a better overall sensitivity for the protonated analytes. The dynamic range was from 10 to 1000 ng ml(-1) from 500 microl of plasma for ABT-518 and the metabolites were detected at levels of the same order of magnitude. Inter-assay accuracies for ABT-518 at five concentration levels were between -9.24 and 6.93% and inter-assay precisions were always <10.7%. Analyte stability was not critical during either storage or processing. This method was successfully applied in a phase I clinical study of ABT-518. The active drug, ABT-518, and all of the metabolites included in the assay could be identified in plasma from dosed patients. We believe that the method described in this paper using an alkaline mobile phase in combination with a basic stable analytical column may also be generally useful for the bioanalysis of other basic drugs.

Robust and cost-effective capillary electrophoresis-mass spectrometry interfaces suitable for combination with on-line analyte preconcentration.

This paper describes several successful cost-effective attempts to couple capillary electrophoresis (CE) and mass spectrometry (MS) without make-up flow or nebulizing gas. An in-depth analysis of several interfaces using conductive spray tips was performed as well as an easy-to-prepare T-junction with direct electrode contact, the latter being the most robust interface. No coating is necessary and the spray voltage is applied through a gold wire positioned at the gap between the separation and spray capillaries. The T-junction interface is made by puncturing a small piece of transparent rubber. The on-line preconcentration CE-MS system allows immunoassay sensitivity, as is demonstrated by a calibration plot in the picomolar range for angiotensin II and gonadorelin. It also shows good reproducibility and has the ability of excellent automation. The secure electrical contact gives a constant spray quality, even with 100% aqueous separation buffers. The described setup has a wide applicability as is demonstrated by the analysis of larger peptides, such as insulin and cytochrome c. Detailed information is given on critical factors in the preparation of the described interfaces.

Capillary electrophoretic bioanalysis of therapeutically active peptides with UV and mass spectrometric detection after on-capillary preconcentration.

An earlier developed capillary electrophoresis (CE) system with an on-capillary adsorptive phase is investigated for its suitability to quantitate low concentrations of angiotensin II and gonadorelin in plasma. An off-line solid-phase extraction is used for sample preparation. The on-line preconcentration CE system allows multiple capillary volumes of sample solution to be injected, increasing the concentration sensitivity of CE with 3-4 orders of magnitude. Furthermore, possible influence of matrix salts can be ruled out by employing a rinsing step after sample application. Using short-wavelength UV detection, reproducibility and linearity in the low nanomolar range were satisfactory. The capillary could be efficiently regenerated using a programmed between-run rinsing procedure, allowing 20-30 large injections of sample extracts. Coating of the capillary improved the robustness of the method. Mass spectrometric detection via a previously reported sheathless interface increased the selectivity and sensitivity substantially. Recommendations are provided for the sample preparation process, the most critical part of the system. Further purification of the sample is required to allow the loading of larger sample volumes and to optimize the system's robustness.

Modification and inhibition of vancomycin group antibiotics by formaldehyde and acetaldehyde.

It is shown that several vancomycin group antibiotics (vancomycin, eremomycin, and avoparcin) undergo spontaneous chemical modifications when kept at room temperature at neutral pH in aqueous solutions containing traces of formaldehyde or acetaldehyde. This chemical modification predominantly results in a mass increase of 12 Da in the reaction with formaldehyde and 26 Da in the case of acetaldehyde. By using tandem mass spectrometry the modification can unambiguously be identified as originating from the formation of a ring-closed 4-imidazolidinone moiety at the N-terminus of the glycopeptide antibiotics, that is, near the receptor binding pocket of the glycopeptide antibiotics. Bioaffinity mass spectrometry shows that this ring-closure results in a dramatically decreased affinity for the peptidoglycan-mimicking D-alanyl-D-alanine receptor. Additionally, in vitro inhibition measurements on two different strains of bacteria have revealed that the modified antibiotics display reduced antibacterial activity. The ring-closure is also shown to have a dissociative effect on the dimerization of the vancomycin-analogue eremomycin. The spontaneous reaction of vancomycin with formaldehyde or acetaldehyde may have implications not only for the clinical use of this class of antibiotics, but also for the effectiveness of these antibiotics when they are used in chiral separation chromatography or capillary electrophoresis.

Metastable ion formation and disparate charge separation in the gas-phase dissection of protein assemblies studied by orthogonal time-of-flight mass spectrometry.

The dissection of specific and nonspecific protein complexes in the gas phase is studied by collisionally activated decomposition. In particular, the gas phase dissection of multiple protonated homodimeric Human Galectin I, E. Coli Glyoxalase I, horse heart cytochrome c, and Hen egg Lysozyme have been investigated. Both the Human Galectin I and E. Coli Glyoxalase I enzymes are biologically active as a dimer, exhibiting molecular weights of approximately 30 kDa. Cytochrome c and Lysozyme are monomers, but may aggregate to some extent at high protein concentrations. The gas phase dissociation of these multiple protonated dimer assemblies does lead to the formation of monomers. The charge distribution over the two concomitant monomers following the dissociation of these multiple protonated dimers is found to be highly dissimilar. There is no evident correlation between the solution phase stability of the dimeric proteins and their gas-phase dissociation pattern. Additionally, in the collisionally activated decomposition spectra diffuse ion signals are observed, which are attributed to monomer ions formed via slow decay of the collisionally activated dimer ions inside the reflectron time-of-flight. Although, the formation of these diffuse metastable ions may complicate the interpretation of collisionally activated decomposition mass spectra, especially when studying noncovalent protein complexes, a simple mathematical equation may be used to reveal their origin and pathway of formation.

Deep brain stimulation for the treatment of Parkinson's disease: subthalamic nucleus versus globus pallidus internus.

OBJECTIVES: Deep brain stimulation of the basal ganglia has become a promising treatment option for patients with Parkinson's disease who have side effects from drugs. Which is the best target-globus pallidus internus (GPi) or subthalamic nucleus (STN)-is still a matter of discussion. The aim of this prospective study is to compare the long term effects of GPi and STN stimulation in patients with severe Parkinson's disease. PATIENTS AND METHODS: Bilateral deep brain stimulators were implanted in the GPi in six patients and in the STN in 12 patients with severe Parkinson's disease. Presurgery and 3, 6, and 12 months postsurgery patients were scored according to the CAPIT protocol. RESULTS: Stimulation of the STN increased best Schwab and England scale score significantly from 62 before surgery to 81 at 12 months after surgery; GPi stimulation did not have an effect on the Schwab and England scale. Stimulation of the GPi reduced dyskinesias directly whereas STN stimulation seemed to reduce dyskinesias by a reduction of medication. Whereas STN stimulation increased the unified Parkinson's disease rating scale (UPDRS) motor score, GPi stimulation did not have a significant effect. Fluctuations were reduced only by STN stimulation and STN stimulation suppressed tremor very effectively. CONCLUSION: Stimulation of the GPi reduces medication side effects, which leads to a better drug tolerance. There was no direct improvement of bradykinesia or tremor by GPi stimulation. Stimulation of the STN ameliorated all parkinsonian symptoms. Daily drug intake was reduced by STN stimulation. The STN is the target of choice for treating patients with severe Parkinson's disease who have side effects from drugs.

Pharmaceutical development of a parenteral lyophilized formulation of the novel antitumor agent aplidine.

Aplidine is a naturally occurring cyclic depsipeptide isolated from the Mediterranean tunicate Aplidium albicans. Aplidine displays promising in vitro and in vivo antitumor activities against various solid human tumor xenografts and is therefore developed now for clinical testing. The aim of this study was to develop a stable parenteral pharmaceutical dosage form for clinical Phase I testing. Aplidine raw material was characterized by using several chromatographic and spectrometric techniques. These experiments showed that aplidine exists as two isomers. A stability-indicating HPLC assay was developed. Solubility testing showed that aplidine exhibits very poor aqueous solubility. Because solubilized aplidine showed substantial degradation under heat and light stress testing conditions, it was decided to develop a lyophilized dosage form. Freeze-drying was carried out with a 500 micrograms/mL solution of aplidine in 40% (v/v) tert-butanol in Water for Injection (WfI) containing 25 mg/mL D-mannitol as a bulking agent. Differential scanning calorimetry was applied to determine the optimal freeze-drying cycle parameters. The prototype, containing 500 micrograms aplidine and 25 mg D-mannitol per vial, was found to be the optimal formulation in terms of solubility, length of lyophilization cycle, and dosage requirements in the forthcoming Phase I clinical studies. Quality control of the freeze-dried formulation demonstrates that the manufacturing process does not affect the integrity of aplidine. The optimal reconstitution solution was found to be 15/15/70% (v/v/v) Cremophor EL/ethanol/WfI (CEW). Both reconstituted product and dilutions of the reconstituted product with normal saline (up to 1:100 v/v) appeared to be stable for at least 24 hours after preparation. Shelf-life data, available thus far, show that the lyophilized formulation is stable for at least 1 year when stored at +2-8 degrees C in the dark.

Stability of thioTEPA and its metabolites, TEPA, monochloroTEPA and thioTEPA-mercapturate, in plasma and urine.

The degradation of N,N',N"-triethylenethiophosphoramide (thioTEPA) and its metabolites N,N',N"-triethylenephosphoramide (TEPA), N, N'-diethylene,N"-2-chloroethylphosphoramide (monochloroTEPA) and thioTEPA-mercapturate in plasma and urine has been investigated. ThioTEPA, TEPA and monochloroTEPA were analyzed using a gas chromatographic (GC) system with selective nitrogen/phosphorous detection; thioTEPA-mercapturate was analyzed on a liquid chromatography-mass spectrometric (LC-MS) system. The influences of pH and temperature on the stability of thioTEPA and its metabolites were studied. An increase in degradation rate was observed with decreasing pH as measured for all studied metabolites. In urine the rate of degradation at 37 degrees C was approximately 2.5+/-1 times higher than at 22 degrees C. At 37 degrees C thioTEPA and TEPA were more stable in plasma than in urine, with half lives ranging from 9-20 h for urine and 13-34 h for plasma at pH 6. Mono- and dichloro derivatives of thioTEPA were formed in urine and the monochloro derivative was found in plasma. Degradation of TEPA in plasma and urine resulted in the formation of monochloroTEPA. During the degradation of TEPA in plasma also the methoxy derivative of TEPA was formed as a consequence of the applied procedure. The monochloro derivative of thioTEPA-mercapturate was formed in urine, whereas for monochloroTEPA no degradation products could be detected.

Electrospray ionization mass spectrometry as a tool to analyze hydrogen/deuterium exchange kinetics of transmembrane peptides in lipid bilayers.

A method is described to study the precise positioning of transmembrane peptides in a phospholipid bilayer combining hydrogen/deuterium (H/D) exchange and nanoelectrospray ionization mass spectrometry. The method was tested by using model systems consisting of designed alpha-helical transmembrane peptides [acetylGW(2)(LA)(5)W(2)Aethanolamine (WALP16) and acetyl-(GA)(3)W(2)(LA)(5)W(2)(AG)(3)ethanolamine (WALP16(+10))] incorporated in large unilamellar vesicles of 1, 2-dimyristoyl-sn-glycero-3-phosphocholine. Both peptides consist of an alternating leucine/alanine hydrophobic core sequence flanked by tryptophan residues as interfacial anchor residues. In the case of WALP16(+10), this sequence is extended at both ends by 5-aa glycine/alanine tails extending into the aqueous phase surrounding the bilayer. H/D exchange of labile hydrogens in these peptides was monitored in time after dilution of the vesicles in buffered deuterium oxide. It was found that the peptides can be measured by direct introduction of the proteoliposome suspension into the mass spectrometer. Several distinct H/D exchange rates were observed (corresponding to half-life values varying from

A search for new metabolites of N,N',N''-triethylenethiophosphoramide.

An attempt was made to unravel the metabolic profile of the alkylating agent N,N',N''-triethylenethiophosphoramide (thioTEPA). thioTEPA and its metabolite N,N',N-triethylenephosphoramide (TEPA) were quantified in urine of treated patients by gas chromatography with selective nitrogen/phosphorous detection. Total alkylating activity was assessed by p-nitrobenzylpyridine reactivity. The total alkylating activity exceeded the amount of thioTEPA and TEPA, indicating the presence of other alkylating metabolites. Solid-phase extraction and liquid-liquid extractions followed by gas chromatography-mass spectrometry analysis revealed the conversion of an aziridinyl function of TEPA into a beta-chloroethyl moiety. This metabolite, N,N'-diethylene-N''-2-chloroethylphosphoramide, was quantified by gas chromatography with selective nitrogen/phosphorous detection and accounted for only 0.69% of the administered dose. Large volumes of urine were concentrated with solid-phase extraction and fractionated with high-performance liquid chromatography. Alkylating activity was determined for each 2-ml fraction and showed the presence of an alkylating compound eluting between 8 and 12 ml. The fractions with alkylating activity were collected, evaporated under a stream of nitrogen at room temperature to dryness, reconstituted in methanol, and subjected to fast atom bombardment-mass spectrometry and fast atom bombardment-tandem mass spectrometry. A new metabolite was found with a molecular mass of 352 Da, the same as that of thioTEPA-mercapturate. thioTEPA-mercapturate is likely the result of glutathione conjugation, after which the glutathione adduct loses two amino acid residues in separate stages. The fragmentation pattern and chromatographic properties of this new metabolite were identical to those of the reference, thioTEPA-mercapturate, which was obtained by incubation of thioTEPA with N-acetylcysteine at pH 11 and 95 degrees C for 30 min. Quantification of thioTEPA-mercapturate was carried out by liquid chromatography-mass spectrometry. The thioTEPA-mercapturate levels in urine accounted for 12.3% of the administered dose and exceeded the amount of TEPA, which was previously assumed to be the main metabolite of thioTEPA. The total excreted amount of thioTEPA and its metabolites accounts for 54-100% of the total alkylating activity, indicating the presence of still other alkylating metabolites.

Covalent and non-covalent dissociations of gas-phase complexes of avoparcin and bacterial receptor mimicking precursor peptides studied by collisionally activated decomposition mass spectrometry.

The gas-phase stability and reactivity of non-covalent complexes of avoparcin and bacterial receptor mimicking precursor peptides were probed by electrospray ionization mass spectrometry combined with collisionally activated decomposition (CAD) studies. The order of the gas-phase stabilities of these non-covalent complexes is different from the order of the stabilities of the same complexes in solution. The specific stereoselectivity observed in non-covalent binding in solution is not retained in the gas phase. The presence of a lysine residue in the bacterial receptor mimicking precursor peptides appears to promote the gas-phase stabilities of the antibiotic-peptide complexes. Complexes of avoparcin with receptor peptides containing a lysine residue are stabilized in the gas phase to such an extent that CAD of these non-covalent complexes proceeds through a competition between non-covalent and covalent fragmentation pathways. These results indicate clearly that the use of CAD mass spectra for the quantitative characterization of the stability of non-covalent complexes in solution should be applied with extreme caution.

Gas phase bimolecular chemistry of isomeric C3H 6Br (+) cations.

The gas phase chemistry of C3H6Br(+) cations generated via low energy electron impact on various dibromopropanes has been studied by using Fourier transform ion cyclotron resonance mass spectrometry. Neutral substrate molecules that have been selected to probe the bimolecular reactivity of the C3H6Br(+) isomers are ammonia, methylamine, trimethylamine, cis-butene, and 2, 3-dimethyl-2-butene. At least three different isomers are characterized on the basis of their different reactivity toward the various substrate molecules. It is suggested that these isomers have (a) the 2-bromo-2-propyl cation structure, (b) the propylenebromomum ion structure, and (c) the cyclic four-membered trimethylenebromonium ion structure. The 2-bromo-2-propyl cations react predominantely via proton transfer. This reaction is hampered for the propylenebromonium ions, which react mainly as electrophiles or bromanyl cation donors. Cyclic trimethylenebromoruum ions react predominantly via adduct formation, even under low pressure conditions, which implies that tturd body collisions are not the only stabilization mechanism.

Echocardiographic diagnosis of effusive-constrictive pericarditis due to staphylococcal pericarditis after cardiac surgery.

Two weeks after coronary artery bypass surgery, a 43-year-old man was readmitted with fever, pneumonia, left pleural effusion, and pericarditis. Echocardiography showed a localized posterior pericardial effusion, pericardial thickening, and bulging of the ventricular septum toward the left ventricle. Right-sided catheterization indicated pericardial constriction. Effusive-constrictive pericarditis was confirmed at surgery. Cardiac imaging played an important role in diagnosis of this unusual complication of cardiac surgery.

Localization of antibody in the central nervous system of a patient with paraneoplastic encephalomyeloneuritis.

We report a patient with severe paraneoplastic encephalomyeloneuritis, occult small-cell carcinoma of the lung, and high titers of circulating antineuronal antibody who died shortly after developing limbic encephalitis. The antibody was of IgG class and reacted specifically with nuclei and cytoplasm of all neurons in the pattern typical for encephalomyelitis and subacute sensory neuropathy associated with small-cell carcinoma (type II, anti-Hu). At autopsy, perivascular inflammatory infiltrates were prominent. All samples of serum, CSF, and postmortem peritoneal and pleural fluid contained high titers of antibody. Direct immunofluorescence of frozen tissue revealed IgG bound to most remaining neurons in multiple brain regions in a pattern similar to indirect immunofluorescence of normal brain tissue. IgG was also bound to tumor. Attempts to elute antibody from tissue decreased background staining but did not remove neuronal immunofluorescence. These results indicate that antibody can access and bind specifically to neuronal antigens in the brain during the course of paraneoplastic disease.