Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths.

The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 1012 /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 1011 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 109 particles.

Clinical potential of methylphenidate in the treatment of cocaine addiction: a review of the current evidence.

BACKGROUND: Cocaine use continues to be a public health problem, yet there is no proven effective pharmacotherapy for cocaine dependence. A promising approach to treating cocaine dependence may be agonist-replacement therapy, which is already used effectively in the treatment of opioid and tobacco dependence. The replacement approach for cocaine dependence posits that administration of a long-acting stimulant medication should normalize the neurochemical and behavioral perturbations resulting from chronic cocaine use. One potential medication to be substituted for cocaine is methylphenidate (MPH), as this stimulant possesses pharmacobehavioral properties similar to those of cocaine. AIM: To provide a qualitative review addressing the rationale for the use of MPH as a cocaine substitute and its clinical potential in the treatment of cocaine dependence. METHODS: We searched MEDLINE for clinical studies using MPH in patients with cocaine abuse/dependence and screened the bibliographies of the articles found for pertinent literature. RESULTS: MPH, like cocaine, increases synaptic dopamine by inhibiting dopamine reuptake. The discriminative properties, reinforcing potential, and subjective effects of MPH and cocaine are almost identical and, importantly, MPH has been found to substitute for cocaine in animals and human volunteers under laboratory conditions. When taken orally in therapeutic doses, its abuse liability, however, appears low, which is especially true for extended-release MPH preparations. Though there are promising data in the literature, mainly from case reports and open-label studies, the results of randomized controlled trials have been disappointing so far and do not corroborate the use of MPH as a substitute for cocaine dependence in patients without attention deficit hyperactivity disorder. CONCLUSION: Clinical studies evaluating MPH substitution for cocaine dependence have provided inconsistent findings. However, the negative findings may be explained by specific study characteristics, among them dosing, duration of treatment, or sample size. This needs to be considered when discussing the potential of MPH as replacement therapy for cocaine dependence. Finally, based on the results, we suggest possible directions for future research.

Peptide affinity chromatography media that bind N(pro) fusion proteins under chaotropic conditions.

To design a generic purification platform and to combine the advantages of fusion protein technology and matrix-assisted refolding, a peptide affinity medium was developed that binds inclusion body-derived N(pro) fusion proteins under chaotropic conditions. Proteins were expressed in Escherichia coli using an expression system comprising the autoprotease N(pro) from Pestivirus, or its engineered mutant called EDDIE, with C-terminally linked target proteins. Upon refolding, the autoprotease became active and cleaved off its fusion partner, forming an authentic N-terminus. Peptide ligands binding to the autoprotease at 4 M urea were screened from a combinatorial peptide library. A group of positive peptides were identified and further refined by mutational analysis. The best binders represent a common motif comprising positively charged and aromatic amino acids, which can be distributed in a random disposition. Mutational analysis showed that exchange of a single amino acid within the peptide ligand caused a total loss of binding activity. Functional affinity media comprising hexa- or octapeptides were synthesized using a 15-atom spacer with terminal sulfhydryl function and site-directed immobilization of peptides derivatized with iodoacetic anhydride. The peptide size was further reduced to dipeptides comprising only one positively charged and one aromatic amino acid. Based on this, affinity media were prepared by immobilization of a poly amino acid comprising lysine or arginine, and tryptophan, phenylalanine, or tyrosine, respectively, in certain ratios. Binding capacities were in the range of 7-15 mg protein mL(-1) of medium, as could be shown for several EDDIE fusion proteins. An efficient protocol for autoproteolytic cleavage using an on-column refolding method was implemented.

Matrix-assisted refolding of autoprotease fusion proteins on an ion exchange column.

Refolding of proteins must be performed under very dilute conditions to overcome the competing aggregation reaction, which has a high reaction order. Refolding on a chromatography column partially prevents formation of the intermediate form prone to aggregation. A chromatographic refolding procedure was developed using an autoprotease fusion protein with the mutant EDDIE from the N(pro) autoprotease of pestivirus. Upon refolding, self-cleavage generates a target peptide with an authentic N-terminus. The refolding process was developed using the basic 1.8-kDa peptide sSNEVi-C fused to the autoprotease EDDIE or the acidic peptide pep6His, applying cation and anion exchange chromatography, respectively. Dissolved inclusion bodies were loaded on cation exchange chromatographic resins (Capto S, POROS HS, Fractogel EMD SO(3)(-), UNOsphere S, SP Sepharose FF, CM Sepharose FF, S Ceramic HyperD F, Toyopearl SP-650, and Toyopearl MegaCap II SP-550EC). A conditioning step was introduced in order to reduce the urea concentration prior to the refolding step. Refolding was initiated by applying an elution buffer containing a high concentration of Tris-HCl plus common refolding additives. The actual refolding process occurred concurrently with the elution step and was completed in the collected fraction. With Capto S, POROS HS, and Fractogel SO(3)(-), refolding could be performed at column loadings of 50mg fusion protein/ml gel, resulting in a final eluate concentration of around 10-15 mg/ml, with refolding and cleavage step yields of around 75%. The overall yield of recovered peptide reached 50%. Similar yields were obtained using the anion exchange system and the pep6His fusion peptide. This chromatographic refolding process allows processing of fusion peptides at a concentration range 10- to 100-fold higher than that observed for common refolding systems.

Yeast cell surface display system for determination of humoral response to active immunization with a monoclonal antibody against EpCAM.

Even though an immunogenic formulation of the murine monoclonal anti-EpCAM (epithelian cell adhesion molecule) antibody Mab 17-1A, has been shown to evoke a strong humoral immune response in both, monkey studies and early clinical trials, conventional anti-EpCAM ELISA could not identify anti-EpCAM immune response in relation to treatment with Mab17-1A. In contrast, usage of cellulose membranes prepared by SPOT technology presenting overlapping EpCAM peptides allowed the unequivocal determination of EpCAM related antibodies present in monkeys sera after immunization with IGN101. Based on such contradictory results, it was of high interest to compare obtained data to a different method for better assessment of their possible interpretation. Therefore, in the present studies, some EpCAM peptides, determined as reactive by binding of IgG isolated from sera of treated monkeys on membranes prepared by SPOT technology, were represented on yeast surface using the pYD1 yeast display vector system. Binding of biotinylated IgG from sera was detected with streptavidin-FITC and quantity of binding was determined by FACS measurement. Though using this completely different method, experiments with pre-immune and immune sera of four monkeys exemplarily are comparable to the results obtained by analysis with synthetic peptide arrays.

Quality of life after multiple trauma: the effect of early onset psychotherapy on quality of life in trauma patients.

BACKGROUND AND AIMS: The aim of this study was to improve health-related quality of life (HRQOL) related to depression, anxiety, pain, physical functioning and social aspects for severely injured trauma survivors by early onset cognitive behavioural therapy applied on the surgical ward. MATERIALS AND METHODS: The study was a randomised, controlled study. Of 298 primary screened patients 171 were eligible and randomised. Ninety-two patients adhered to follow-up investigations at 6 and 12 months. Main outcome measure was a sum score according to O'Brien calculated of five different questionnaires (BDI, SF-36, STAI, SCL 90R, F-SOZU-22). RESULTS: The sum score for overall HRQOL did not show significant group differences at follow-up. Effects on HRQOL sub-dimensions within groups have been found. In the dimension of depression therapy group showed significant improvement from the first measurement to discharge from hospital (p < 0.001), 6 MFU (p = 0.004) and to 12 MFU (p = 0.013). Measures of anxiety showed significant improvement for the therapy group at discharge from hospital (p = 0.001). In the control group there was only a significant reduction in depression and anxiety from surgical ward to discharge (p = 0.013/p = 0.031). CONCLUSIONS: Early onset cognitive therapy is not effective in improving overall HRQOL of severely injured patients but shows promising effects on depression and anxiety up to 12 months after trauma.

Faecal steroid metabolites for non-invasive assessment of reproduction in common warthogs (Phacochoerus africanus), red river hogs (Potamochoerus porcus) and babirusa (Babyrousa babyrussa).

The objectives of this study were to analyse faecal steroid metabolites in African and South East Asian pig species kept in European zoos. Species studied were the warthog (Phacochoerus africanus), the red river hog (Potamochoerus porcus) and the babirusa (Babyrousa babyrussa). Faecal samples were collected 1-3 times per week from non-pregnant and pregnant captive female warthogs (n = 9), red river hogs (n = 7) and babirusas (n = 5). Enzyme-immunoassays for faecal progesterone, androgen, and oestrogen metabolites, were tested for their ability to determine follicular and luteal phases. In all three species, oestrous cycles could be monitored with 20alpha-OH- and 20-oxo-pregnane assays. In contrast, oestrogens and androgens were not useful in characterising follicular activity during the oestrous cycle in any species. Faecal 20alpha-OH- and 20-oxo-pregnane values were significantly correlated. Faecal pregnane concentrations revealed species-specific differences. Luteal phase values of 20alpha-OH-pregnanes were considerably higher than 20-oxo-pregnanes; 20alpha-OH-pregnanes were in the range of 3-10 microg/g in warthogs and red river hogs, whereas concentrations were 30-200 microg/g faeces in the babirusa. Regular oestrus cycles had a length of about 35 days in all three species studied. Results indicated a seasonal influence on the occurrence of reproductive cycles in the warthog with anoestrous periods in the European summer. The red river hog was found to be a seasonal and poly oestrous breeder; oestrus cycles started by January and continued until summer. In contrast, the babirusa showed non-seasonal ovarian cyclicity. In pregnant red river hogs, progesterone metabolites were comparable to luteal phase values of the oestrous cycle during the first 3 months of gestation, but did further increase during the last month of pregnancy. Oestrogens and 17-oxo-androstanes were significantly elevated during the second half of gestation. In summary, the reproductive biology of three exotic pig species was studied using non-invasive faecal steroid analysis and these methods were used for comparative investigations of oestrous cycles, pregnancy and seasonality.

Directed immobilization of peptide ligands to accessible pore sites by conjugation with a placeholder molecule.

When small ligands are immobilized onto a porous chromatography medium, only a limited number of binding sites contributes to the interaction with the target molecule. The main part of the ligand molecules is distributed on sites that are not accessible for the target protein due to steric hindrance. To direct the ligand into a well-accessible position, the ligand was conjugated to a large molecule that acted as a placeholder during the immobilization step. Then the placeholder molecule was cleaved off and washed out. Two linear peptides with affinity for lysozyme and human blood coagulation factor VIII, respectively, were studied as model systems. The protected peptide ligand was covalently linked to a 20-kDa poly(ethylene glycol) molecule containing an acid-labile linker. After selective deprotection of the peptide and purification, immobilization of this conjugate on a preactivated chromatography matrix was performed alternatively through the free N-terminus, the epsilon-amino group of lysine, or the sulfohydryl group of cysteine. After the immobilization reaction, the spacer molecule and remaining protecting groups were cleaved off and the gels were tested by affinity chromatography. This novel immobilization technique substantially increased the binding capacity and the ligand utilization for the target protein, and site-specific immobilization could be demonstrated.

Direct synthesis of peptides on convective interaction media monolithic columns for affinity chromatography.

Solid-phase peptide synthesis was performed on glycidyle methacrylate-co-ethylene dimethacrylate monoliths using Fmoc chemistry. The native epoxy groups were amino-functionalized by reaction with ethylenediamine or ammonia ions. A peptide directed against human blood coagulation factor VIII was synthesized as a model peptide. Amino acid analysis revealed the correct amino acid ratio as present in the sequence. The ligand density of 5 micromol/mL was equal to that achieved with conventional peptide immobilization via epoxy groups. These supports were directly used as peptide affinity chromatography matrixes. The functionality of the CIM monolithic supports was proven by affinity chromatography of factor VIII. The ammonia-functionalized support performed with low hydrophobicity and did not show unspecific adsorption of proteins.

Quality of Life after traumatic brain injury: A systematic review of the literature.

In modern industrial countries traumatic brain injury (TBI) is a common sequel after different kinds of accidents especially amongst young male adults. Apart from medical and economic consequences Quality of Life (QoL) after TBI becomes increasingly important in outcome assessment. Besides the classical domains of QoL (physical, psychological, social) cognitive impairments are playing an important role especially for TBI patients. In 1991 the Meran conference set important standards and formulated basic guidelines for defining and measuring QoL in surgery, but a special index for TBI patients has not yet been developed. Instead, QoL research concentrates on physical, medical, psychological and social problems only. Based on the existing QoL concept extended by the cognitive aspect it was the aim of this review to give an overview about the recent QoL research in TBI patients since 1991. Sixteen studies in TBI patients mentioning at least 2 domains of QoL (physical, psychological, social, cognitive) were published since 1991. Five of them considered all 4 domains of QoL. All studies except of one dealt with psychological and social problems. Only half of the studies considered cognitive impairments. Four studies tried to define QoL, but none of them included the cognitive component. There was no consensus regarding the definition and the choice of measurement instrument for QoL after TBI. This review of 16 studies considering outcome and QoL after TBI confirms that a homogenous and clinically relevant QoL concept for this group of patients is still missing. Further research in TBI patients should include all 4 domains of QoL.