Elimination of factor VIII-specific B cells by immunotoxins composed of a single factor VIII domain fused to Pseudomonas exotoxin A.

Essentials There is still a need for novel therapeutic approaches for hemophilia A patients with inhibitors. A factor VIII domain was used as the targeting moiety for elimination of FVIII-specific B cells. The immunodominant C2 domain was fused to exotoxin A from Pseudomonas aeruginosa (hC2-ETA). Murine C2 domain-specific B cells were selectively and efficiently eliminated by hC2-ETA ex vivo. SUMMARY: Background Today, the most serious complication for patients with hemophilia A undergoing factor VIII (FVIII) replacement therapy is the development of neutralizing antibodies (inhibitors). Although inhibitors can be eradicated by application of high doses of FVIII, the immune tolerance induction therapy fails in up to 30% of patients. Hence, there is still an urgent need for novel therapeutic approaches for patients with persisting inhibitors. Objectives In the present study, the potential use of immunotoxins containing exotoxin A (ETA) from Pseudomonas aeruginosa for selective elimination of FVIII-specific B cells was explored. Methods The immunodominant C2 domain of human FVIII was used as a targeting moiety instead of the full-length FVIII protein and the resulting human C2 domain-ETA fusion protein (hC2-ETA) was produced in Escherichia coli. Results Binding studies with monoclonal C2 domain-specific antibodies confirmed the conformational integrity of the C2 domain in hC2-ETA. The functionality of hC2-ETA was tested ex vivo by incubation of splenocytes from inhibitor-positive FVIII knockout mice with hC2-ETA and controls. FVIII-specific memory B cells from splenocytes were differentiated by FVIII stimulation in antibody-secreting cells (ASC) and detected by an enzyme-linked immunospot assay. Although the controls showed no effect, incubation of splenocytes with hC2-ETA reduced the number of C2-specific ASC in a dose-dependent fashion, indicating specific and efficient elimination of C2-specific memory B cells. Conclusions Overall, the results of the study support the fact that FVIII domain immunotoxins might be a potential new tool for the elimination of FVIII-specific B cells in patients with hemophilia A and persisting inhibitors.

Anti-factor VIII antibodies in brothers with haemophilia A share similar characteristics.

INTRODUCTION: The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) is currently the most serious complication for patients with haemophilia A undergoing FVIII replacement therapy. Several genetic factors have been acknowledged as risk factors for inhibitor development. AIM: To analyze the influence of genetic factors on the nature of the humoral immune response to FVIII in eight brother pairs with inhibitors. METHODS: The domain specificity of FVIII-specific IgG was analysed by antibody binding to FVIII fragments and homologue-scanning mutagenesis (HSM). The FVIII-specific IgG subclasses were measured by direct ELISA. RESULTS: Of the 16 patient analysed with both methods, 12 had A2- and 13 had C2-specific IgG. The presence of A1-, A3- or C1-specific IgG was identified in nine of 14 patients analysed by HSM. IgG1, IgG2 and IgG4 subclasses contributed to the anti-FVIII IgG response, and the amount of FVIII-specific IgG1 (r = 0.66) and IgG4 (r = 0.69) correlated significantly with inhibitor titres. Patients with high concentrations of total anti-FVIII IgG (r = 0.69) or high inhibitor titres (r = 0.52) had a high proportion of FVIII-specific IgG4. Statistical analysis revealed trends/evidence that the subclass distribution (P = 0.0847) and domain specificity to HC/LC (P = 0.0883) and A2/C2 (P = 0.0011) of anti-FVIII IgG were more similar in brothers compared to unrelated subjects. CONCLUSION: Overall, our data provide a first hint that anti-FVIII IgG characteristics are comparable among haemophilic brothers with inhibitors. Whether genetic factors also influence the nature of patients' antibodies needs to be confirmed in a larger study population.

Long-Term Outcomes of ADHD: A Systematic Review of Self-Esteem and Social Function.

OBJECTIVE: To compare the long-term self-esteem and social function outcomes of individuals with untreated and treated ADHD across childhood, adolescence, and adulthood. METHOD: A systematic search of 12 databases was performed to identify peer-reviewed, primary research articles, published January 1980 to December 2011, reporting long-term self-esteem and/or social function outcomes (≥2 years; life consequences distinct from symptoms) of individuals with untreated or treated ADHD. RESULTS: Overall, 127 studies reported 150 outcomes. Most outcomes were poorer in individuals with untreated ADHD versus non-ADHD controls (57% [13/23] for self-esteem; 73% [52/71] for social function). A beneficial response to treatment (pharmacological, nonpharmacological, and multimodal treatments) was reported for the majority of self-esteem (89% [8/9]) and social function (77% [17/22]) outcomes. CONCLUSION: Untreated ADHD was associated with poorer long-term self-esteem and social function outcomes compared with non-ADHD controls. Treatment for ADHD was associated with improvement in outcomes; however, further long-term outcome studies are needed.

Predicted effect size of lisdexamfetamine treatment of attention deficit/hyperactivity disorder (ADHD) in European adults: Estimates based on indirect analysis using a systematic review and meta-regression analysis.

BACKGROUND: There are few approved therapies for adults with attention-deficit/hyperactivity disorder (ADHD) in Europe. Lisdexamfetamine (LDX) is an effective treatment for ADHD; however, no clinical trials examining the efficacy of LDX specifically in European adults have been conducted. Therefore, to estimate the efficacy of LDX in European adults we performed a meta-regression of existing clinical data. METHODS: A systematic review identified US- and Europe-based randomized efficacy trials of LDX, atomoxetine (ATX), or osmotic-release oral system methylphenidate (OROS-MPH) in children/adolescents and adults. A meta-regression model was then fitted to the published/calculated effect sizes (Cohen's d) using medication, geographical location, and age group as predictors. The LDX effect size in European adults was extrapolated from the fitted model. Sensitivity analyses performed included using adult-only studies and adding studies with placebo designs other than a standard pill-placebo design. RESULTS: Twenty-two of 2832 identified articles met inclusion criteria. The model-estimated effect size of LDX for European adults was 1.070 (95% confidence interval: 0.738, 1.401), larger than the 0.8 threshold for large effect sizes. The overall model fit was adequate (80%) and stable in the sensitivity analyses. CONCLUSION: This model predicts that LDX may have a large treatment effect size in European adults with ADHD.

Energy costs & performance of transtibial amputees & non-amputees during walking & running.

This study compared energy costs and performance differences of walking and running for transtibial amputee (TTA) and matched non-amputee runners. TTA were tested with 3 prosthetic feet: traditional foot, SACH; general purpose, energy storing and return (ESAR) foot, Renegade; running-specific ESAR foot, Nitro. During walking, VO2 and gait efficiency (GE) were similar between prosthetic feet. VO2 was increased (21-33%) and GE was decreased for TTA compared to controls. Self-selected walking speed (SSWS) was slower for SACH (4-6%) compared to Renegade and Nitro but SSWS for TTA was slower (16-22%) than controls. During running, VO2 was increased (8-18%) and GE was decreased using SACH and Renegade, compared to Nitro. During running, VO2 was greater (9-38%), GE was decreased and SSRS was slower (17-30%) for TTA, than controls. VO2 peak was similar for controls and TTA using Nitro, but peak running speed was slower for TTA. In conclusion, during walking energy costs are mostly similar between prosthetic feet, but ESAR feet likely provide faster SSWS for TTA. During running, energy costs and performance are improved for TTA using Nitro. Nonetheless, for all prosthetic feet conditions, TTA demonstrated an energy cost and performance disadvantage during walking and running compared to non-amputee runners.

Selection and characterisation of FVIII-specific single chain variable fragments.

The development of inhibitory anti-FVIII antibodies is currently the most severe complication in the treatment of haemophilia A patients. Inhibitor eradication can be achieved by immune tolerance induction (ITI). Recent findings suggest a correlation between the FVIII-specific IgG subclass distribution and the duration or outcome of ITI. To quantify FVIII-specific IgG subclasses in patients' plasma FVIII-specific IgG standards are required. Here, the isolation of FVIII-specific single chain variable fragments (scFvs) from synthetic phage display libraries and the characterisation of their FVIII domain specificity are described. The isolated scFv 1G10, which binds to the FVIII A2 domain, was cloned into the context of the four human IgG (hIgG) subclasses and expressed in mammalian cells. Purified 1G10-hIgG1, -hIgG2, -hIgG3 and -hIgG4 are used as standards to determine the absolute amounts and relative contribution of the different FVIII-specific IgG subclasses in future studies. The results from these studies will eventually add to understanding the role of the FVIII-specific IgG subclass distribution as prognostic factor for the outcome of ITI.

Metabolic demands of rock climbing in transfemoral amputees.

This pilot study compared the energy expenditure required to climb an indoor rock wall, in amputees utilizing five prosthetic configurations. Three experienced climbers (1M age 21 yr, 2F ages 30 and 49 yr) with unilateral transfemoral amputation climbed a 9.14 m indoor rock wall, 5.9 Yosemite Decimal Scale rating, using the following prosthetic configurations: 1. no prosthesis; 2. stubby prosthesis-foot forward; 3. stubby prosthesis-foot backward; 4. articulated prosthesis-knee unlocked; 5. articulated prosthesis-knee locked. Subjects climbed three times with each configuration resulting in 15 climbs per subject. Metabolic data was collected using the COSMED K4b(2) system. VO(2) was 15, 18 and 20% greater in the articulated unlocked condition (mean+/-SE: 20.5+/-0.8 ml.kg (-1).min (-1)), and 11, 13 and 15% greater in the articulated locked condition (19.7+/-0.9 ml.kg (-1).min (-1)), compared to the no prosthesis (17.8+/-0.7 ml.kg (-1).min (-1)), stubby backward (17.4+/-0.7 ml.kg (-1).min (-1)) and stubby forward (17.1+/-0.9 ml.kg (-1).min (-1)) conditions. Participants expended 11-20% more energy using the articulated prostheses than with the stubby and no prosthesis conditions. In persons with transfemoral amputation, use of an articulated prosthesis in indoor rock climbing may be a disadvantage in many aspects including competition, training, rehabilitation and satisfaction with the activity.

Bioaccumulation of trace metals in the Antarctic amphipod Orchomene plebs: evaluation of toxicokinetic models.

Bioaccumulation of Cd, Co, Cu, Ni, Pb and Zn in Antarctic gammaridean amphipod collectives, Orchomene plebs (Hurley, 1965), was investigated during a cruise of RV "Polarstern" to the Wedell Sea. With the sole exception of Cd the organisms accumulated metals during exposure and depurated them in uncontaminated seawater. Four independent toxicokinetic experiments and one field study were modelled simultaneously to calculate the following size-dependent bioconcentration factors for organisms with body length 10 mm (BCF(10 mm)): 130 (Co), 4030 (Cu), 190 (Ni), 2900 (Pb), and 5210 (Zn). On the time scale of our experiments the data suggest an increased metal uptake by previously exposed test organisms. The collectives investigated may be regarded as potentially suitable biomonitors for Co, Cu, Ni, Pb and Zn but not for Cd. An approach to evaluate the sensitivity of Orchomene plebs as a biomonitor of waterborne metals in the field indicates minimal increments of the ambient exposure concentrations of 0.01 microg Co l(-1), 0.2 microg Cu l(-1), 0.4 microg Ni l(-1), 0.6 microg Pb l(-1) and 0.3 microg Zn l(-1).

Trace metals in Antarctic copepods from the Weddell Sea (Antarctica).

The concentrations of Cd, Co, Cu, Ni, Pb and Zn were determined in the Antarctic copepods Rhincalanus gigas (Brady, 1883), Calanus propinquus (Brady, 1883), Calanoides acutus (Giesbrecht, 1902), Metridia curticauda (Giesbrecht, 1889) and Metridia gerlachei (Giesbrecht, 1902). Samples were taken at seven different stations between 18.01.1999 and 19.02.1999. Metal concentrations in biological tissue were determined by graphite furnace atomic absorption spectroscopy (GFAAS) with Zeeman background correction and by flame AAS (air-acetylene) with deuterium background correction. We found high mean Cd concentrations in the Metridia species of about 10 microg Cd g(-1) and 3-6 microg Cd g(-1) in the other copepods. Co and Pb concentrations were low in all species investigated (<0.1 microg Co g(-1) and <1 microg Pb g(-1)). Zn concentrations were high in M. gerlachei and R. gigas (518 and 430 microg Zn g(-1)). In comparison to copepods from Arctic Seas (Fram Strait, Greenland Sea) and the North Sea, Cd and Cu concentrations appear higher in Antarctic copepods, while Ni and Pb concentrations are similar in both polar regions and Pb concentrations are higher in the North Sea. Variability between species and different regions are discussed.

Bioaccumulation of trace metals in the calanoid copepod Metridia gerlachei from the Weddell Sea (Antarctica).

Bioaccumulation of Cd, Co, Cu, Ni, Pb and Zn in the Antarctic calanoid copepod Metridia gerlachei (Giesbrecht 1902) was investigated during a cruise of RV 'Polarstern' to the Weddell Sea, primarily to provide information on accumulation strategies for the metals tested. With the sole exception of Cd, the copepod accumulated metals during exposure and depurated them in uncontaminated seawater. The process of uptake and depuration was successfully described by a hyperbolic model, leading to significant estimations of the following experimental bioconcentration factors (BCFs): 210 (Co), 3430 (Cu), 3060 (Ni), 670 (Pb) and 2090 (Zn). Furthermore, we provide an approach to evaluate the sensitivity of Metridia gerlachei as a biomonitor of water-borne metals in the field; the results indicate minimal increments in ambient exposure concentrations of: 0.5 microg Cu l(-1), 0.8 microg Ni l(-1), 0.6 microg Pb l(-1) and 0.2 microg Zn l(-1), suggesting a high sensitivity of M. gerlachei for biomonitoring.

Bioaccumulation of trace metals in the copepod Calanoides acutus from the Weddell Sea (Antarctica): comparison of two-compartment and hyperbolic toxicokinetic models.

Bioaccumulation of Cd, Co, Cu, Ni, Pb and Zn in the Antarctic calanoid copepod Calanoides acutus (Giesbrecht, 1902) was investigated during a cruise of RV 'Polarstern' to the Weddell Sea. Main goals were to provide information on accumulation strategies of the organisms tested and to verify toxicokinetic models as a predictive tool. Except for Cd, the organisms accumulated metals upon exposure. It was possible to estimate significant model parameters of two-compartment and hyperbolic models. These models were successfully verified in a second toxicokinetic uptake study. The model verification was extended in a third uptake study with increasing external metal dosing. We found a linear increase of net uptake with external waterborne metal exposures up to 80 microg Pb l(-1), with excellent predictions of the two-compartment model. For Co both models give reasonable predictions up to 20 microg Co l(-1). Regarding Cu, Ni and Zn only hyperbolic model predictions were in good agreement with measured values up to 150 microg Cu l(-1), 80 microg Ni l(-1) and 290 microg Zn l(-1). Due to a decrease of Cd body burden in the experiments, only the hyperbolic model was applicable, leading to reliable predictions up to 20 microg Cd l(-1). These concentrations largely determine the range for which these models may serve as a predictive tool.

L-2-amino-4-phosphonobutanoic acid and 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid reduce paired-pulse depression recorded from medial perforant path in the dentate gyrus of rat hippocampal slices.

The perforant path is the major excitatory cortical projection to the hippocampal dentate gyrus. Field potentials from the medial perforant path exhibit paired-pulse depression when evoked at interstimulus intervals of 40 to 800 msec. We found that an early component of paired-pulse depression recorded at interstimulus intervals of 40 to 100 msec from slices of rat hippocampus was reduced by L-2-amino-4-phosphonobutanoic acid (L-AP4) (20 microM) without a change in the size of the first field potential in the pair. Paired-pulse depression evoked at intervals of 200 to 800 msec was not reduced. 1S,3R-1-Aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), DL-2-amino-3-phosphonopropionic acid and carbachol also reduced paired-pulse depression in a manner similar to L-AP4. Picrotoxin, phaclofen, theophylline or atropine did not reduce paired-pulse depression. Furthermore, paired-pulse depression (40-100 msec) does not appear to involve glutamate uptake or N-methyl-D-aspartate receptors as L-alpha-aminoadipate did not alter paired-pulse depression and neither trans-L-pyrrolidine-2,4-dicarboxylate and L-alpha-aminoadipate nor D-2-amino-5-phosphonopropionic acid blocked the effect of L-AP4 on paired-pulse depression. 4-Aminopyridine inhibits a potassium current that has a similar time course to the L-AP4-induced reduction of paired-pulse depression, however, paired-pulse depression was increased with exposure to 4-aminopyridine. These results indicate that the mechanism underlying paired-pulse depression consists of two components, the early component being reduced by L-AP4, 1S,3R-ACPD, DL-2-amino-3-phosphonopropionic acid and carbachol.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased sensitivity to adenosine in the rat dentate gyrus molecular layer two weeks after partial entorhinal lesions.

The molecular layer of the dentate gyrus exhibits extensive circuit and receptor reorganization after entorhinal lesions and in Alzheimer's disease, including decreased adenosine (A1) receptor binding in the terminal zone of damaged perforant path fibers. We examined the adenosine-sensitivity of evoked synaptic activity recorded from the rat dentate gyrus molecular layer in hippocampal slices prepared after electrolytic lesions were placed in approximately the middle third of the entorhinal cortex. Extracellular field potentials (EFPs) recorded in slices prepared from animals two days post-lesion were small, upward-going, and exhibited paired-pulse potentiation, but by two weeks post-lesion EFPs had recovered to large, downward-going responses that exhibited paired-pulsed depression. EFPs recorded from two week post-lesion slices were about 2-fold more sensitive (P < or = 0.05) to exposure to adenosine when compared to EFPs recorded from slices from unlesioned animals. Adenosine-induced reduction of paired-pulse depression was similar between unlesioned and post-lesion slices. AChE histochemistry performed after recording revealed dense staining in the dentate gyrus molecular layer of post-lesion slices as compared to slices from unlesioned animals, confirming that sprouting of cholinergic fibers occurred as expected from previous entorhinal lesion studies. Autoradiography performed on adjacent slices showed a decrease in binding to A1-adenosine receptors in the dentate gyrus molecular layer in post-lesion slices as compared to slices from unlesioned animals, indicating that there was a loss of presynaptically located A1-adenosine receptors on damaged perforant pathway terminals. These results indicate that, in addition to the recovery of the major excitatory signal to the hippocampus after entorhinal cell loss, this signal is more sensitive to modulation by adenosine, suggesting an increase in A1-adenosine receptor efficacy in the reinnervated region.

Adenosine, L-AP4, and baclofen modulation of paired-pulse potentiation in the dentate gyrus: interstimulus interval-dependent pharmacology.

Paired-pulse potentiation of the glutamate-mediated excitatory postsynaptic potential (EPSP) recorded in the dentate gyrus molecular layer is thought to be mediated presynaptically. It is known that the activation of adenosine (A1) and GABAB receptors results in the reduction of glutamate release in the dentate molecular layer via presynaptic mechanisms. To examine possible modulatory roles of these receptors on paired-pulse potentiation, we examined the effects of adenosine and baclofen (a GABAB agonist) on paired-pulse potentiation using extracellular recording from the lateral perforant path in rat hippocampal slices maintained in vitro. We compared these effects with those of L-alpha-amino-4-phosphonobutyric acid (L-AP4) over a wide range of interstimulus intervals (ISIs). L-AP4 enhanced paired-pulse potentiation over the full range of ISIs tested (40-800 ms), whereas adenosine enhanced paired-pulse potentiation only at ISIs of 40-100 ms. In contrast, baclofen reduced paired-pulse potentiation only at ISIs of 400-800 ms. Furthermore, baclofen increased the amplitude of lateral perforant path field potentials, previously reported to be baclofen-insensitive. These results suggest that paired-pulse potentiation can be modulated through the activation of adenosine and baclofen receptors, indicate that this modulation is dependent on ISI, and show that there are at least two pharmacologically separable components of paired-pulse potentiation in the dentate gyrus.

Axon sprouting in the rodent and Alzheimer's disease brain: a reactivation of developmental mechanisms?

Research over the past 15 years has led to a comprehensive description of the processes of axonal sprouting and synaptic reorganization in the hippocampus. Previous studies on axonal sprouting have now been supplemented with recent studies on excitatory amino acid receptor plasticity. These and related studies pave the way to research strategies which detail the molecular mechanisms of the sprouting response. The re-expression of the fetal form of alpha-tubulin mRNA in rat after entorhinal lesions was found to be similar to the re-expression of the human fetal form of alpha-tubulin in Alzheimer's brain. This result suggests that the sprouting process may involve a reactivation of certain developmental mechanisms and that this may possibly contribute to the etiology of Alzheimer's disease.

Structure-function studies on N-oxalyl-diamino-dicarboxylic acids and excitatory amino acid receptors: evidence that beta-L-ODAP is a selective non-NMDA agonist.

Excitatory amino acids and their receptors play an important role in both normal synaptic transmission and excitotoxic-mediated neuronal death. In the present investigation we have prepared a series of glutamate analogs and examined the pharmacological specificity with which they interact with excitatory amino acid receptors. Included within this group of compounds is a potent excitotoxic amino acid, beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (beta-L-ODAP). This excitotoxin is of particular interest because it has been identified as a major causative agent of human neurolathyrism, a disease characterized by permanent spastic paralysis. The site of action of beta-L-ODAP was delineated with both electrophysiological recordings in hippocampal slices and radioligand binding assays in synaptic plasma membranes. We report that beta-L-ODAP is a potent agonist at the non-N-methyl-D-aspartate (NMDA) type of excitatory amino acid receptor. beta-L-ODAP interacts most potently with the quisqualate class of non-NMDA receptors (IC50 = 1.3 microM), less potently with the kainate receptor (IC50 = 17 microM), and very weakly with NMDA receptors. The specificity of this binding was consistent with physiological experiments that demonstrated that beta-L-ODAP-induced depolarizations were potently blocked by the newly identified non-NMDA receptor antagonist, CNQX, but were not affected by the NMDA antagonist D-AP5. These results extend recent studies that have focused on the contribution of NMDA receptors to excitotoxicity and highlight the potential involvement of non-NMDA receptors in excitotoxic-mediated cell death.

Carbachol depresses synaptic responses in the medial but not the lateral perforant path.

The effects of applications of carbachol on evoked synaptic responses recorded in the dentate gyrus of guinea pig hippocampal slices were examined. Carbachol depressed potentials recorded extracellularly in the medial perforant path terminal zone, but did not significantly alter field potentials recorded in the lateral perforant path terminal zone. Carbachol-induced depression was reversed by applications of the muscarinic antagonists, atropine or pirenzepine. It was suggested that the difference observed in carbachol-induced depression of medial versus lateral perforant path field potentials may be due to regional differences in acetylcholine receptor distribution in the molecular layer of the dentate gyrus.

Use of a self-generating percoll gradient and single cell cytotoxicity assay to identify tumor-lytic properties of inflammatory neutrophils.

Within a murine model of regional immunotherapy, the cytolytic potential of peritoneal neutrophils could not be confirmed or quantified using routine techniques of cell separation and chromium release assays. We, therefore, developed procedures for the enrichment of neutrophils and estimation of the frequency of killer cells. Peritoneal exudate cells from mice injected with Corynebacterium parvum were fractionated on a self-generating Percoll gradient to enrich for neutrophils and deplete macrophages. A significant enrichment of neutrophils (greater than 90%) was obtained in a band corresponding to a density of 1.088 with a recovery of 35-50% of input. Neutrophil-enriched cell populations were then mixed with tumor cells to examine neutrophil-target interactions at the single cell level. Conjugates of neutrophils and tumor targets were obtained and the majority were lytic. With the aid of trypan blue staining and safranin counterstaining, it was possible to distinguish effector cells from targets and neutrophils from other host cells. The frequency of conjugates was dependent upon the effector to target cell ratio and was not affected by changes in temperature (range 4-30 degrees C). The post-binding lytic events were initiated rapidly after conjugation and tumor lysis was completed within 30 min. The lytic events occurred optimally between 25 degrees and 37 degrees C. The present studies support the role of neutrophils in tumor lysis following administration of an immunoadjuvant. The techniques described are important to further study the role of neutrophils in disease states as well as the underlying mechanisms of neutrophil-mediated tumor cytotoxicity.