Topical resiquimod dosing regimens in patients with multiple actinic keratoses: a multicentre, partly placebo-controlled, double-blind clinical trial.

BACKGROUND: Topical immune response modifiers are established for actinic keratosis (AK) treatment and efforts are underway to make further improvements to their efficacy and safety. OBJECTIVES: To investigate the optimal dosing regimens of the Toll-like receptor 7/8 agonist resiquimod in terms of efficacy, safety and tolerability. METHODS: In a multicentre, partly placebo-controlled, double-blind clinical trial, we randomized 217 patients with AK lesions to 0·03% resiquimod gel once-daily application three times per week for 4 weeks or seven times within 2 weeks or five times for 1 week (arms 1/2/3) followed by a treatment-free interval of 8 weeks and one repetition of the cycle. In two additional arms (arms 4/5), patients applied either resiquimod gel 0·01% or 0·03% three times per week up to a biological end point defined by skin erosion or for a maximum duration of 8 weeks. Clearance was assessed clinically and histologically. RESULTS: Complete clinical clearance ranged from 56% to 85% with the highest rate observed in arm 2. Resiquimod 0·03% gel was more effective than 0·01% gel. Clearance rates in arms 1/2/3 were comparable and higher than with placebo and were reached with 24, 14 and 10 gel applications, respectively. Overall, 128 patients (59%) experienced treatment-related adverse reactions. CONCLUSIONS: Resiquimod 0·03% gel is more effective than 0·01% gel. From the perspectives of safety and tolerability, the lower concentration and shorter duration are preferable. The clinical response in arms 2/3 was reached with fewer gel applications. The dosing regimens that used the biological end point (arms 4/5) proved equally efficacious as predefined treatment durations and may therefore be suitable for personalized AK treatment.

BASALIT trial: double-blind placebo-controlled allergen immunotherapy with rBet v 1-FV in birch-related soya allergy.

BACKGROUND: Conflicting results exist on the effect of allergen immunotherapy (AIT) on pollen-related food allergy. We aimed to investigate the efficacy of one-year AIT with the folding variant (FV) of recombinant (r) Bet v 1 on birch-related soya allergy. METHODS: Of 138 subjects with Bet v 1 sensitization, 82 were positive at double-blind placebo-controlled food challenge (DBPCFC) with soya. A total of 56 of 82 were randomized in the ratio of 2:1 (active: placebo). Per-protocol population (PPP) had received ≥150 µg of allergen or placebo preparation. OUTCOME MEASURES: lowest observed adverse effect levels (LOAEL), postinterventional occurrence of objective signs (objS) at any dose level, sIgE/IgG4 against Bet v 1 and Gly m 4. Between-group changes were investigated (ancova, Mann-Whitney U-test, Fisher exact test). RESULTS: Baseline characteristics including LOAELs were comparable in both groups with objS and subjS occurring in 82% and 95% of active (n = 38) vs 78% and 83% of placebo group (n = 18). After AIT, objS occurred in 24% and 47%, respectively. LOAEL group differences showed a beneficial tendency (P = 0.081) for LOAELobjective in PPP (30 active, 15 placebo). sIgG4 raised only in active group (Bet v 1: P = 0.054, Gly m 4: P = 0.037), and no relevant changes occurred for sIgE. Only 56% of the intended sample size was recruited. CONCLUSION: For the first time, we present data on the effect of rBet v 1-FV on birch-related soya allergy. rBet v 1-FV AIT induced significant immunogenic effects. Clinical assessment showed a tendency in favour of the active group but did not reach statistical significance.

Selective adsorption in ion exchange membranes: The effect of solution ion composition on ion partitioning.

Electrodialysis is a water desalination technology that enables selective separation of ions, making it a promising solution for sustainable water reuse. The selectivity of the process is mainly determined by the properties of ion exchange membranes that can vary depending on the composition of ions in water, such as water uptake and charge density. In this work, we studied selective adsorption of Na+ and K+ ions in various ion exchange membranes considering the effect of solution ion composition on membrane water volume fraction. For that purpose, we conducted membrane adsorption experiments using solutions with Na+ and K+ ions with different ion compositions including Li+, Ca2+ or Mg2+ ions at different concentrations (0.001 - 0.25 M). The experiments showed that with the total ion concentration and the amount of divalent ions in solution, the membrane water volume fraction decreases while the selective adsorption of the smaller (hydrated) K+ ions over the Na+ ions in the membrane increases. We developed a theoretical framework based on Boublik-Mansoori-Carnahan-Starling-Leland (BMCSL) theory to describe the effect of membrane water volume fraction on selective adsorption of the ions by including volumetric effects, such as size exclusion. The developed framework was used to describe ion partitioning results of the membrane adsorption experiments. In addition, the effect of solution ion composition on selective ion removal during electrodialysis operation was evaluated using experimental data and theoretical calculations. The results of this study show that considering volumetric effects can improve the ion partitioning description in ion exchange membranes for solutions with various ion compositions.

Long-term (6 and 12 months) follow-up of two prospective, randomized, controlled phase III trials of photodynamic therapy with BF-200 ALA and methyl aminolaevulinate for the treatment of actinic keratosis.

BACKGROUND: Two phase III trials of photodynamic therapy (PDT) with BF-200 ALA, a recently approved nanoemulsion formulation of 5-aminolaevulinic acid (ALA) demonstrated high clearance rates in mild-to-moderate actinic keratosis (AK). The comparison to a registered methyl aminolaevulinate (MAL) cream demonstrated significantly superior total patient clearance rates. OBJECTIVES: To evaluate long-term efficacy and safety of PDT for AK 6 and 12 months after the last PDT with BF-200 ALA, MAL or placebo. METHODS: The follow-up phase (FUP) was performed with patients of two phase III studies. Both studies compared BF-200 ALA with placebo, one of the studies additionally with MAL. Overall recurrence rates and various subgroups (light source, lesion severity, lesion location, complete responders after first PDT) were assessed 6 and 12 months after the last PDT. RESULTS: Recurrence rates were similar for BF-200 ALA and MAL, with a tendency to lower recurrence rates for BF-200 ALA. The proportion of patients who were fully cleared during PDT and remained completely clear for at least 12 months after PDT were 47% for BF-200 ALA (both studies) and 36% for MAL treatment. The subgroup that was illuminated with narrow wavelength LED lamps reached 69% and 53% for BF-200 ALA (both studies, respectively) and 41% for MAL. No safety concerns were reported. CONCLUSIONS: The FUP data confirmed the high efficacy and safety of PDT with BF-200 ALA. The slightly lower recurrence rates after BF-200 ALA treatment compared with MAL treatment enhanced the better treatment outcome due to the significantly superior efficacy.

Small-polaron based holograms in LiNbO3 in the visible spectrum.

Diffraction efficiency, relaxation behavior and dependence on pump-beam intensity of small-polaron based holograms are studied in thermally reduced, nominally undoped lithium niobate in the visible spectrum (λ = 488 nm). The pronounced phase gratings with diffraction efficiency up to η = (10.8 ± 1.0)% appeared upon irradiation by single ns-laser pulses (λ = 532 nm) and are comprehensively assigned to the optical formation of spatially modulated densities of small bound NbLi4+ electron polarons, NbLi4+:NbNb4+ electron bipolarons, and O⁻ hole polarons. A remarkable quadratic dependence on the pump-beam intensity is discovered for the recording configuration K || c-axis and can be explained by the electro-optic contribution of the optically generated small bound polarons. We discuss the build-up of local space-charge fields via small-polaron based bulk photovoltaic currents.

Photodynamic therapy with BF-200 ALA for the treatment of actinic keratosis: results of a multicentre, randomized, observer-blind phase III study in comparison with a registered methyl-5-aminolaevulinate cream and placebo.

BACKGROUND: Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) or its methylester [methyl-5-aminolaevulinate (MAL) or 5-amino-4-oxopentanoate] was recently ranked as first-line therapy for the treatment of actinic keratosis (AK) and is an accepted therapeutic option for the treatment of neoplastic skin diseases. BF-200 ALA (Biofrontera Bioscience GmbH, Leverkusen, Germany) is a gel formulation of ALA with nanoemulsion for the treatment of AK which overcomes previous problems of ALA instability and improves skin penetration. OBJECTIVES: To evaluate the efficacy and safety of PDT of AKs with BF-200 ALA in comparison with a registered MAL cream and with placebo. METHODS: The study was performed as a randomized, multicentre, observer-blind, placebo-controlled, interindividual trial with BF-200 ALA, a registered MAL cream and placebo in a ratio of 3:3:1. Six hundred patients, each with four to eight mild to moderate AK lesions on the face and/or the bald scalp, were enrolled in 26 study centres in Germany, Austria and Switzerland. Patients received one PDT. If residual lesions remained at 3months after treatment, PDT was repeated. RESULTS: PDT with BF-200 ALA was superior to placebo PDT with respect to patient complete clearance rate (78·2% vs. 17·1%; P<0·0001) and lesion complete clearance rate (90·4% vs. 37·1%) at 3months after the last PDT. Moreover, superiority was demonstrated over the MAL cream regarding the primary endpoint patient complete clearance (78·2% vs. 64·2%; P<0·05). Significant differences in the patient and lesion complete clearance rates and severity of treatment-related adverse events were observed for the narrow- and broad-spectrum light sources. CONCLUSIONS: BF-200 ALA is a very effective, well-tolerated new formulation for AK treatment with PDT and is superior to a registered MAL medication. Efficacies and adverse events vary greatly with the different light sources used.

Hologram recording via spatial density modulation of Nb(Li)(4+/5+) antisites in lithium niobate.

Hologram recording is studied in thermally reduced, nominally undoped lithium niobate in the time domain from 10 ns to 100 s by means of intense ns pump laser pulses (λ = 532 nm) and continuous-wave probe light (λ = 785 nm). It is shown that mixed absorption and phase gratings can be recorded within 8 ns that feature diffraction efficiencies up to 23 % with non-exponential relaxation and lifetimes in the ms-regime. The results are explained comprehensively in the frame of the optical generation of a spatial density modulation of Nb(Li)(4+/5+) antisites and the related optical features, i.e. absorption as well as index changes mutually related via the Kramers-Kronig-relation. Implications of our findings, such as the electrooptical properties of small bound Nb(Li)(4+) polarons, the optical features of Nb(Li)(4+):Nb(Nb)(4+) bipolarons, Nb(Nb)(4+) free polarons and O-hole-polarons, the impact of light polarization of pump and probe beams as well as of the polaron density are discussed.

Effects of feed composition on the fouling on cation-exchange membranes desalinating polymer-flooding produced water.

HYPOTHESIS: Cation exchange membranes (CEMs) are subject to fouling when utilized to desalinate wastewater from the oil and gas industry, hampering their performance. The kind and extent of the fouling are most likely dependent on the composition of the stream, which in practical applications can vary significantly. EXPERIMENTS: Fouling experiments were performed on commercial cation exchange membranes, which were used in electrodialysis runs to desalinate solutions of varying composition. The variations included ionic strength, type of ions, amount of viscosifying polyelectrolyte (partially hydrolyzed polyacrylamide), presence of crude oil, and surfactants. Performance parameters, like electric potential and pH, were monitored during the runs, after which the membranes were recovered and analyzed. FINDINGS: Fouling was detected on most CEMs and occurred mainly in the presence of the viscosifying polyelectrolyte. Under normal pH conditions (pH ~ 8), the polyelectrolyte fouled the concentrate side of the CEMs, as expected due to electrophoresis. However, by applying a current in the opposite direction, the polyelectrolyte layer could be removed. Precipitation occurred mostly on the opposite side of the membrane, with different morphology depending on the feed composition.

Long-term follow-up of photodynamic therapy with a self-adhesive 5-aminolaevulinic acid patch: 12 months data.

BACKGROUND: Photodynamic therapy with a self-adhesive 5-aminolaevulinic acid (5-ALA) patch shows high efficacy rates in the treatment of mild to moderate actinic keratosis (AK) in short term trials. OBJECTIVES: The purpose of the trial was to follow up patients after successful 5-ALA patch-PDT at 3 month intervals over a total period of 12 months. Patients who had received placebo-PDT or cryosurgery served for comparison. PATIENTS/METHODS: Three months after therapy, 360 patients from two separate randomized parallel group phase III studies (one superiority trial vs. placebo-PDT, one noninferiority trial vs. cryosurgery) were suitable for the follow-up study. Patients had to show at least one successfully treated AK lesion after initial therapy. A total of 316 patients completed the follow-up. RESULTS: Twelve months after a single treatment, 5-ALA patch-PDT still proved superior to placebo-PDT and cryosurgery (P < 0.001 for all tests). On a lesion basis, efficacy rates were 63% and 79% for PDT, 63% for cryosurgery and 9% and 25% for placebo-PDT. Recurrence rates of patch-PDT proved superior to those of cryosurgery (per protocol set: P = 0.011, full analysis set: P = 0.049). While 31% of cryosurgery lesions were still hypopigmented after 1 year, the 5-ALA patch-PDT groups showed hypopigmentation in 0% (superiority trial) and 3% (noninferiority trial) of the treated lesions. CONCLUSION: Twelve months after a single 5-ALA patch-PDT the majority of lesions were still cleared with an excellent cosmetic outcome. 5-ALA patch-PDT proved to be superior to cryosurgery in the noninferiority study setting.

Optimization of photodynamic therapy with a novel self-adhesive 5-aminolaevulinic acid patch: results of two randomized controlled phase III studies.

BACKGROUND: Photodynamic therapy (PDT) is increasingly used for treatment of actinic keratoses (AKs) but is a cumbersome procedure. A thin self-adhesive patch (PD P 506 A) containing 5-aminolaevulinic acid (5-ALA) was developed to facilitate PDT. OBJECTIVES: To investigate efficacy and safety of the patch in comparison with placebo-PDT (superiority design, observer-blinded; study AK 03) and standard therapy, cryosurgery (noninferiority design, open; study AK 04). METHODS: Two separate confirmatory randomized parallel-group phase III studies were set up. In total, 449 patients with up to eight mild to moderate AK study lesions located on the head were treated in 29 German study centres (study AK 03: 103 patients; study AK 04: 346 patients). RESULTS: Twelve weeks after treatment, 5-ALA patch-PDT proved to be superior to placebo-PDT (P < 0.001) and cryosurgery (P = 0.007). Efficacy rates on a lesion basis were 82% (AK 03) and 89% (AK 04) for PDT, 77% for cryosurgery and 19% (AK 03) and 29% (AK 04) for placebo-PDT. Local reactions at the treatment site occurred in almost all patients treated with 5-ALA patch-PDT or cryosurgery. Headache was the only side-effect not related to the treatment site which occurred in more than one patient. CONCLUSIONS: PD P 506 A is an innovative, easy-to-handle 5-ALA patch for PDT of mild to moderate AK lesions. Compared with current PDT procedures, pretreatment (e.g. curettage) is not needed and handling is considerably facilitated. A single PDT treatment results in efficacy rates being statistically significantly superior to placebo and cryosurgery.

Influence of solution composition on fouling of anion exchange membranes desalinating polymer-flooding produced water.

HYPOTHESIS: Anion exchange membranes (AEMS) are particularly prone to fouling when employed to desalinate polymer flooding produced water (PFPW), an abundant sub-product from the oil and gas industry. The formation of fouling on an AEM will be affected by the composition of the solution, which includes various dissolved salts, partially hydrolyzed polyacrylamide (HPAM), crude oil, and surfactants. EXPERIMENTS: Electrodialysis experiments were performed to desalinate feed solutions with different compositions, aiming to distinguish between their individual and combined effects. The solutions contained diverse mono- and divalent ions. The analysis included data collected during the desalination and characterization of the fouled AEMs by diverse analytical techniques. FINDINGS: HPAM produced the most severe effects in terms of visible fouling and increase of resistance. This polyelectrolyte fouls the AEM by adsorbing on its surface and by forming a viscous gel layer that hampers the replenishment of ions from the bulk solution. Ca and Mg have a large influence on the formation of thick HPAM gel layers, while the oily compounds have only a minimal influence acting mainly as a destabilizing agent. The membranes also presented scaling consisting of calcium precipitates. The effects of the gel layer were minimized by applying current reversal and foulant-free solution.

Anaerobic methanethiol degradation and methanogenic community analysis in an alkaline (pH 10) biological process for liquefied petroleum gas desulfurization.

Anaerobic methanethiol (MT) degradation by mesophilic (30 degrees C) alkaliphilic (pH 10) communities was studied in a lab-scale Upflow Anaerobic Sludge Bed (UASB) reactor inoculated with a mixture of sediments from the Wadden Sea (The Netherlands), Soap Lake (Central Washington), and Russian soda lakes. MT degradation started after 32 days of incubation. During the first 252 days, complete degradation was achieved till a volumetric loading rate of 7.5 mmol MT/L/day, and sulfide, methane, and carbon dioxide were the main reaction products. Temporary inhibition of MT degradation occurred after MT peak loads and in the presence of dimethyl disulfide (DMDS), which is the autooxidation product of MT. From day 252 onwards, methanol was dosed to the reactor as co-substrate at a loading rate of 3-6 mmol/L/day to stimulate growth of methylotrophic methanogens. Methanol was completely degraded and also a complete MT degradation was achieved till a volumetric loading rate of 13 mmol MT/L/day (0.77 mmol MT/gVSS/day). However, from day 354 till the end of the experimental run (day 365), acetate was formed and MT was not completely degraded anymore, indicating that methanol-degrading homoacetogenic bacteria had partially outcompeted the methanogenic MT-degrading archea. The archeal community in the reactor sludge was analyzed by DGGE and sequencing of 16S rRNA genes. The methanogenic archea responsible for the degradation of MT in the reactor were related to Methanolobus oregonensis. A pure culture, named strain SODA, was obtained by serial dilutions in medium containing both trimethyl amine and dimethyl sulfide (DMS). Strain SODA degraded MT, DMS, trimethyl amine, and methanol. Flow sheet simulations revealed that for sufficient MT removal from liquefied petroleum gas, the extraction and biological degradation process should be operated above pH 9.

Sublingual immunotherapy in pollen-induced seasonal rhinitis and conjunctivitis: a randomized controlled trial.

BACKGROUND: Sublingual immunotherapy (SLIT) is a recognized and safe treatment for allergic rhinitis and conjunctivitis. The aim was to evaluate the efficacy and safety of tablets for grass and rye pollen- induced rhinitis and conjunctivitis. METHODS: A double-blind, randomized, placebo-controlled trial was carried out over 9 months. 105 patients received a standardized grass/rye mix extract or a placebo using sublingual drops during the build-up phase. Drops were replaced by sublingual tablets during the maintenance phase (300 IR/daily). RESULTS: In patients that received active treatment, a significantly lower total symptom score (rhinitis and conjunctivitis) compared to the placebo group was observed (p = 0.038). The investigators' assessment revealed a significant improvement in favor of the active treatment group (p = 0.018). Skin reactivity to grass and rye pollen was significantly reduced in the active treatment group (p < 0.05). No statistical difference was observed between the two groups for serum-specific IgG4 levels. Side effects were local and mild, and no severe systemic reactions were reported. CONCLUSION: This study indicates that tablet-based sublingual immunotherapy was safe and significantly improved grass/rye pollen-induced rhinoconjunctivitis symptoms. It was also associated with a significant inhibition of the immediate skin response.

Molecular identification of the TCA alloantigen as the transferrin receptor on T-cells of a leukemic patient with serum from a monozygotic twin brother.

The transferrin receptor is encoded by a diallelic locus which had been described as the T-cell antigenic system TCA. Using a human lymphoblastoid T-cell line (HSB) which reportedly carries a transferrin receptor and which we showed to be TCA-2 positive by serology, it appeared that only a TCA-2 but not a TCA-1 specific alloantiserum had the capacity to precipitate a protein with a molecular weight of 94,000, which comigrated exactly with the Mr 94,000 protein precipitated by the rabbit anti-HSB and rabbit anti-SB antisera, as well as the transferrin receptor which was precipitated by a transferrin-antitransferrin complex. The positive sera and the latter complex were shown to cross-react in a binding assay. Conversely, a monospecific TCA-1 alloantiserum was shown to react with the Mr 94,000 protein of TCA-1-positive leukocyte suspensions. The allelomorphism of the transferrin receptor may be relevant for the eventual regulatory mechanism controlling cell proliferation, which would be essential at least for those cells that are supposed to play an immunological role like the TCA-positive cells which are described as T-cells probably involved in the process of immunosuppression. In addition, we found increasing amounts of autoantibodies with TCA-1 specificity in serum from a healthy individual (referred to as "donor" in this paper), whose brother at the same time suffered a chronic myeloid leukemia, probably associated with a retrovirus, and whose blood in contrast was shown to contain cytotoxic anti-TCB, from a diallelic locus expressed on theophylline-insensitive T-cells, and anti-B-cell activity.

Synthesis of a viral protein with molecular weight of 30,000 (p30) by leukemic cells and antibodies cross-reacting with Simian sarcoma virus p30 in serum of a chronic myeloid leukemia patient.

We studied a patient with a Philadelphia chromosome-positive chronic myeloid leukemia, who died in relapse after multiple transfusions and grafting with bone marrow from his monozygotic twin brother (referred to as "donor" in this paper). We present data indicating that this patient may have had a retro-virus infection that this virus is related to the group of exogenous primate type C retroviruses. Antibodies to simian sarcoma virus (SSV) M.W. 30,000 protein (p30) but not endogenous feline virus RD-114 could be found in patient but not donor serum. Patient but not donor cells were able to actively synthesize a p30 protein that could be precipitated with patient serum and rabbit anti-SSV p30 but not with donor serum or rabbit anti-RD 114 p30. Patient p30 resembles SSV p30 but not RD-114 p30 in peptide mapping by limited proteolysis and subsequent slab gel electrophoresis. Patient but not donor cells were able to actively synthesize a M.W. 78,000 protein that could be precipitated with goat anti-SSV. An enzyme with properties of reverse transcriptase was increased 30-fold ion patient cells when compared with donor and other control cells. Related to the presence of widespread infectious agents may be the finding that, in the course of the patient's disease, donor serum showed increasing amounts of possibly immunoregulatory (Cancer Research, submitted for publication) antibodies, reactive with autologous and, more effectively, with patient-derived cell membrane M. W. 80,000 protein (a possible idiotypic receptor structure) and M.W. 94,000 protein (a T-cell alloantigen).

Standardization of double blind placebo controlled food challenge with soy within a multicentre trial.

BACKGROUND: Multicentre trials investigating food allergies by double blind placebo controlled food challenges (DBPCFC) need standardized procedures, challenge meals and evaluation criteria. We aimed at developing a standardized approach for identifying patients with birch related soy allergy by means of DBPCFC to soy, including determination of threshold levels, in a multicentre setting. METHODS: Microbiologically stable soy challenge meals were composed of protein isolate with consistent Gly m 4 levels. Patients sensitized to main birch allergen Bet v 1 and concomitant sensitization to its soy homologue Gly m 4 underwent DBPCFC. Outcome was defined according to presence and/or absence of ten objective signs and intensity of eight subjective symptoms as measured by visual analogue scale (VAS). RESULTS: 138 adult subjects (63.8% female, mean age 38 years) underwent DBPCFC. Challenge meals and defined evaluation criteria showed good applicability in all centres involved. 45.7% presented with objective signs and 65.2% with subjective symptoms at soy challenge. Placebo challenge meals elicited non-cardiovascular objective signs in 11.6%. In 82 (59.4%) subjects DBPCFC was judged as positive. 70.7% of DPBCFC+ showed objective signs and 85.4% subjective symptoms at soy challenge. Subjective symptoms to soy challenge meal in DBPCFC+ subjects started at significantly lower dose levels than objective signs (p < 0.001). Median cumulative eliciting doses for first objective signs in DBPCFC+ subjects were 4.7 g [0.7-24.7] and 0.7 g [0.2-4.7] total soy protein for first subjective symptoms (p = 0.01). CONCLUSIONS: We present the hitherto largest group of adults with Bet v 1 and Gly m 4 sensitization being investigated by DBPCFC. In this type of food allergy evaluation of DBPCFC outcome should not only include monitoring of objective signs but also scoring of subjective symptoms. Our data may contribute to standardize DBPCFC in pollen-related food allergy in multicentre settings. TRIAL REGISTRATION: EudraCT: 2009-011737-27.

Plasmids useable as gene-cloning vectors in an in vitro packaging by coliphage lambda: "cosmids".

A plasmid which contains a cos site of lambda and can be packaged into lambda bacteriophage particles is termed a "cosmid". Such plasmids can be used as gene cloning vectors in conjunction with an in vitro packaging system. The properties of a new series of cosmids based on the ColE1 replicon are described, including small temperature-sensitive plasmids which have lost mobilisation functions and carry no IS sequences. Amongst these plasmids are vectors for XmaI, BglII, BamHI, HindIII, PstI, KpnI, SalI and EcoRI. It is demonstrated that by using cosmids in particular size ranges these plasmids provide a high efficiency cloning system which yields essentially only hybrid clones without resort to a second selection or screening step, and without prior modification (e.g. phosphatase) treatment of the DNA. Attempts were made to optimise the cloning properties of the cosmid system. An Escherichia coli "gene bank" was obtained with an efficiency of 5 . 10(5) clones per microgram of E. coli DNA, and in which any particular unselected marker may be found in about one out of every 400 clones. It was demonstrated that deletion of mobilisation functions leads to loss of ability to form relaxation-complex without affecting copy number or segregation properties of the temperature-sensitive derivatives. The vectors are amplifiable in chloramphenicol to make up about 50% of the total cellular DNA.

A mouse monoclonal antibody detecting a DR-related MT2-like specificity: serology and biochemistry.

A mouse monoclonal antibody (7.3.19.1) was produced which reacts with class II molecules on B cells and monocytes of DR3, DR5, and/or DRw6 positive donors only. Using this moab and two others, three different groups of class II molecules could be identified. Furthermore, a differential precipitation pattern was found which correlates with a DR-related variable expression of the MT2-like polymorphic determinants on the cell surface. Addition of 7.3.19.1 to MLCs did not result in significant inhibition in controls to the two other moabs tested. Normal CTL activity was found in such a stimulated responder population.

Identification of a new lymphocyte subset surface antigen, the expression of which disappears after in vitro and in vivo stimulation. Distribution, biochemistry, and functional studies.

Two monoclonal antibodies (MoAbs) are described (MD 2.6, IgG1 and MD 4.3, IgG2a) that react with a nonlineage specific lymphocyte subset surface antigen. This antigen is expressed on B cells, a subset of both T8+ and T4+ cells, cells that exert killer and natural killer cell activity in vitro, B cells in lymph nodes, and a small percentage of thymocytes. Expression of the antigen was found to be variable on T cells but not on B cells among individuals. Following polyclonal activation, expression of the determinant detected was lost from the cell surface. Both MD+ and MD+ cells responded to PHA and in MLC. MLC resulted in the generation of cytotoxic T lymphocytes and primed T lymphocytes in both the MD+ and MD+ subpopulations. In contrast, the response to soluble antigens was found to reside almost exclusively in the MD-subset. Immunoprecipitation indicates that the MoAbs react with an antigen that has a molecular weight of 220-240 KD which can be cleaved into subunits of 70-80 kD by beta-mercaptoethanol.

Topography and characteristics of specific binding sites for non-opioid gamma-type endorphins in the rat brain as studied by autoradiography with [35S]Met-desenkephalin-gamma-endorphin.

An in vitro autoradiographic study was performed to characterize specific rat brain binding sites for non-opioid neuroleptic-like gamma-type endorphins, using [35S]Met-des-enkephalin-gamma-endorphin ([35S]Met-DE gamma E; [35]S-beta-endorphins(5-17)) with high specific activity as radioligand. The binding sites appeared to be confined to rat forebrain regions, e.g., orbital cortex, frontal cortex, cingulate cortex, piriform cortex, nucleus accumbens, amygdala, mediodorsal nucleus of the thalamus and arcuate and periventricular nuclei of the hypothalamus. These regions are part of the mesocorticolimbic feedback circuit. Densitometric analysis of the autoradiographs revealed that the density of the binding sites was highest in the mediodorsal nucleus of the thalamus and the amygdala. Concentration-dependent displacement of [35S]Met-DE gamma E (500 pM) with DE gamma E yielded an IC50 of 0.6 nM whereas DE alpha E (beta-endorphin(6-16)) had an IC50 of 210 nM. Various endorphins, sharing the gamma-endorphin C terminus, displaced [35S]Met-DE gamma E to the same extent as non-labelled DE gamma E (at 10(-6) M) whereas non-endorphin peptides did not show displacing capacity. Possible relationships of the binding sites with opioid receptors were investigated. DAMGO (mu) and DPDPE (delta) displaced [35S]Met-DE gamma E to some extent at 10(-6) M whereas U69,593 (kappa) was inactive, suggesting that the binding sites for gamma-type endorphins may resemble mu- and delta-opioid receptors in some aspects. Similarly, relationships with dopamine receptors were investigated. Haloperidol partially displaced [35S]Met-DE gamma E whereas sulpiride, SKF38,393 and 3-PPP at 10(-6) M did not induce significant displacement. Thus, binding sites are distinct from dopamine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)