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Understanding species differences in the placental transfer of monoclonal antibodies is important to inform species selection for nonclinical safety assessment, interpret embryo-fetal changes observed in these studies, and extrapolate their human relevance. Data presented here for a fully human immunoglobulin G2 monoclonal antibody (IgG2X) revealed that, during organogenesis, in both the cynomolgus monkey (gestation day 35 [gd35]) and the rat (gd10) the extent of IgG2X placental transfer (approximately 0.5% maternal plasma concentration, MPC) was similar to the limited published human data for endogenous IgG. At this early gestational stage, IgG2X placental transfer was approximately 6-fold higher in the rabbit (gd10). By the end of organogenesis, rat embryonic plasma concentrations (gd16) exceeded those in the cynomolgus monkey (gd50) by approximately 3-fold. These data suggest that relative to the cynomolgus monkey, the rabbit (and to a lesser extent the rat) may overestimate potential harmful effects to the human embryo during this critical period of development. Beyond organogenesis, fetal IgG2X plasma concentrations increased approximately 10-fold early in the second trimester (gd50-70) in the cynomolgus monkey and remained relatively unchanged thereafter (at approximately 5% MPC). Late gestational assessment was precluded in rabbits due to immunogenicity, but in rats, fetal IgG2X plasma concentrations increased more than 6-fold from gd16 to gd21 (reaching approximately 15% MPC). In rats, maternal exposure consistent with that achieved by ICH S6(R1) high-dose selection criteria resulted in embryonic plasma concentrations, reaching pharmacologically relevant levels during organogenesis. Furthermore, dose proportional exposure in both mothers and embryos indicated that this was unlikely to occur at the lower therapeutic dose levels used in humans., (© 2014 Wiley Periodicals, Inc.)

NFAT transcription factors control the proliferation and survival of peripheral lymphocytes. We have reported previously that the short isoform NFATc1/αA whose generation is induced by immune receptor stimulation supports the proliferation and inhibits the activation-induced cell death of peripheral T and B cells. We will show in this study that in novel bacterial artificial chromosome transgenic mice that express EGFP under the control of entire Nfatc1 locus the Nfatc1/Egfp transgene is expressed as early as in double-negative thymocytes and in nonstimulated peripheral T and B cells. Upon immune receptor stimulation, Nfatc1/Egfp expression is elevated in B, Th1, and Th2 cells, but only weakly in T regulatory, Th9, and Th17 cells in vitro whose generation is affected by TGFβ. In naive lymphocytes, persistent immune receptor signals led to a 3-5 increase in NFATc1/αA RNA levels during primary and secondary stimulation, but a much stronger induction was observed at the protein level. Whereas anti-CD3(+)CD28 stimulation of primary T cells induces both NFATc1/αA and their proliferation and survival, anti-IgM stimulation of B cells induces NFATc1/αA and proliferation, but activation-induced cell death after 3-d incubation in vitro. The anti-IgM-mediated activation-induced cell death induction of B cells in vitro is suppressed by anti-CD40-, LPS-, and CpG-mediated signals. In addition to inducing NF-κB factors, together with anti-IgM, these signals also support the generation of NFATc1/αA. According to these data and the architecture of its promoter region, the Nfatc1 gene resembles a primary response gene whose induction is affected at the posttranscriptional level.

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.

Using two clinical samples of patients, the presented studies examined the construct validity of the recently revised Anxiety Sensitivity Index-3 (ASI-3). Confirmatory factor analyses established a clear three-factor structure that corresponds to the postulated subdivision of the construct into correlated somatic, social, and cognitive components. Participants with different primary clinical diagnoses differed from each other on the ASI-3 subscales in theoretically meaningful ways. Specifically, the ASI-3 successfully discriminated patients with anxiety disorders from patients with nonanxiety disorders. Moreover, patients with panic disorder or agoraphobia manifested more somatic concerns than patients with other anxiety disorders and patients with nonanxiety disorders. Finally, correlations of the ASI-3 scales with other measures of clinical symptoms and negative affect substantiated convergent and discriminant validity. Substantial positive correlations were found between the ASI-3 Somatic Concerns and body vigilance, between Social Concerns and fear of negative evaluation and socially inhibited behavior, and between Cognitive Concerns and depression symptoms, anxiety, fear of negative evaluation, and subjective complaints. Moreover, Social Concerns correlated negatively with dominant and intrusive behavior. Results are discussed with respect to the contribution of the ASI-3 to the assessment of anxiety-related disorders.

Dispositional styles of coping with threat influence memory for threatening information. In particular, sensitizers excel over repressors in their memory for threatening information after long retention intervals, but not after short ones. We therefore suggested that sensitizers, but not repressors, employ active maintenance processes during the retention interval to selectively retain threatening material. Sensitive maintenance was studied in 2 experiments in which participants were briefly exposed to threatening and nonthreatening pictures (Experiment 1, N = 128) or words (Experiment 2, N = 145). Following, we administered unannounced recognition tests before and after an intervening task that generated either high or low cognitive load, assuming that high cognitive load would impede sensitizers' memory maintenance of threatening material. Supporting our hypotheses, the same pattern of results was obtained in both experiments: Under low cognitive load, sensitizers forgot less threat material than repressors did; no such differences were observed under high cognitive load.

The Borna disease virus (BDV) nucleoprotein (N) monomer resembles the nucleoprotein structures from rabies virus (RABV) and vesicular stomatitis virus (VSV). We show that BDV N assembles into ring- and string-like structures in the presence of 5' genomic BDV RNA. RNA induced polymerization is partly RNA-specific since polymerization is inefficient in the presence of 3' genomic BDV RNA or E. coli RNA. Mutagenesis of basic residues located in the cleft made up by the N- and C-terminal domains of N abrogate RNA-induced polymerization indicating that BDV N binds RNA similarly as observed in case of RABV and VSV N-RNA complexes. Bound RNA is not protected and sensitive to degradation. N-RNA polymers form complexes with the phosphoprotein P as required for functional transcription or replication units. Our data indicate that BDV N utilizes similar structural principles for N-RNA and N-P-RNA complex formation as observed for related negative strand RNA viruses., (Copyright 2009 Elsevier Inc. All rights reserved.)

Purpose: Many of the antiepileptic drugs (AED) used in therapy of temporal lobe epilepsy (TLE) are known as cytochrome P450 (CYP, P450) inducers. These AEDs are thought to modulate androgen and estrogen pathways in hippocampus, and therefore cause mental and reproductive disorders found in TLE patients. In the present study, we analyzed expression of androgen receptor (AR), estrogen receptor alpha (ERalpha), and CYP3A in the hippocampus of TLE patients and in murine hippocampal cell line HN25.1., Methods: Patients and cell lines had been treated with P450-inducing or noninducing AEDs, or with prednisolone, applied to prevent oedema formation prior to neurosurgical resection of the epileptic hippocampus. Human patient samples were analyzed by immunohistochemical approach, the HN25.1 cell line by quantitative RT-PCR, CAT reporter gene assay, and immunoblot., Results: In both, humans and cell lines, the expression of testosterone metabolising CYP3A4 (human) or CYP3A11 (mouse) and AR was up-regulated when P450-inducing AEDs and/or prednisolone had been applied. AR responsive CAT reporter gene assay indicated an increase of AR-signalling after treatment of the HN25.1 cells with the P450-inducers phenytoin and carbamazepine. ERalpha expression was increased only by the P450-inducing AEDs, but not by prednisolone, which indicates that pathways different from CYP3A4/11 led to ERalpha enhancement., Discussion: We conclude that P450-inducing AEDs influence AR expression and signalling in hippocampus most likely via CYP3A4/11-induction. The HN25.1 cell line holds promise to investigate the correlation between drug application and AR regulation, and to specifically address issues that are relevant to human TLE patients.

Mitochondria play central roles in energy homeostasis, metabolism, signaling, and apoptosis. Accordingly, the abundance, morphology, and functional properties of mitochondria are finely tuned to meet cell-specific energetic, metabolic, and signaling demands. This tuning is largely achieved at the level of transcriptional regulation. A highly interconnected network of transcription factors regulates a broad set of nuclear genes encoding mitochondrial proteins, including those that control replication and transcription of the mitochondrial genome. The same transcriptional network senses cues relaying cellular energy status, nutrient availability, and the physiological state of the organism and enables short- and long-term adaptive responses, resulting in adjustments to mitochondrial function and mitochondrial biogenesis. Mitochondrial dysfunction is associated with many human diseases. Characterization of the transcriptional mechanisms that regulate mitochondrial biogenesis and function can offer insights into possible therapeutic interventions aimed at modulating mitochondrial function.

Energy depletion during ischemia leads to disturbed ionic homeostasis and accumulation of neuroactive substances in the extracellular space, subsequently leading to volume changes in astrocytes. Confocal microscopy combined with 3D reconstruction was used to quantify ischemia-induced astrocyte volume changes in cortical slices of GFAP/EGFP transgenic mice. Twenty-minutes of oxygen-glucose deprivation (OGD) or oxygen-glucose deprivation combined with acidification (OGD(pH 6.8)) revealed the presence of two distinct astrocytic populations, the first showing a large volume increase (HR astrocytes) and the second displaying a small volume increase (LR astrocytes). In addition, changes in resting membrane potential (V(m)), measured by the patch-clamp technique, supported the existence of two astrocytic populations responding differently to ischemia. Although one group markedly depolarized during OGD or OGD(pH 6.8), only small changes in V(m) toward more negative values were observed in the second group. Conversely, acidification (ACF(pH 6.8)) led to a uniform volume decrease in all astrocytes, accompanied by only a small depolarization. Interestingly, two differently responding populations were not detected during acidification. Differences in the expression of inwardly rectifying potassium channels (Kir4.1), glial fibrillary acidic protein (GFAP), and taurine levels in cortical astrocytes were detected using immunohistochemical methods. We conclude that two distinct populations of astrocytes are present in the cortex of GFAP/EGFP mice, based on volume and V(m) changes during exposure to OGD or OGD(pH 6.8). Immunohistochemical analysis suggests that the diverse expression of Kir4.1 channels and GFAP as well as differences in the accumulation of taurine might contribute to the distinct ability of astrocytes to regulate their volume., (2008 Wiley-Liss, Inc.)

Although the gastrointestinal tract is a rich source of melatonin and possesses numerous melatonin-binding sites, the role of melatonin in this tissue has not yet been fully elucidated. In this work we focused on the role of melatonin in the modulation of ion transport in rat distal colon. Whereas melatonin had no effect on colonic secretion or caused only infrequent and small changes in the short circuit current (Isc) due to its solvent ethanol, this mediator significantly modulated the secretion elicited by some secretagogues. Out of the five substances tested (prostaglandin E(2); 5-hydroxytryptamine; bethanechol; histamine; sodium nitroprusside) melatonin inhibited the effect of prostaglandin E(2) (PGE(2)) and sodium nitroprusside (SNP). Melatonin concentration-dependently decreased PGE(2)-evoked Isc and this inhibitory effect was more obvious from the mucosal side. The basal level of cAMP in colonic mucosa was not influenced by melatonin, but this drug prevented a PGE(2)-induced increase in the level of cAMP. The neurotoxin tetrodotoxin blocked the inhibitory effect of melatonin on SNP-induced Isc. Our data suggests that melatonin takes part in the modulation of colonic ion transport. The modulatory effect of melatonin on PGE(2)-induced Isc occurs directly at the level of the epithelium, whereas the effect on SNP-induced Isc is indirect and located in tetrodotoxin-sensitive enteric neurons.

Peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator-1alpha (PGC-1alpha) is a highly regulated transcriptional coactivator that coordinates energy metabolism in mammals. Misregulation of PGC-1alpha has been implicated in the pathogenesis of several human diseases, including diabetes, obesity, and neurological disorders. We identified SCF(Cdc4) as an E3 ubiquitin ligase that regulates PGC-1alpha through ubiquitin-mediated proteolysis. PGC-1alpha contains two Cdc4 phosphodegrons that bind Cdc4 when phosphorylated by Glycogen Synthase Kinase 3beta (GSK3beta) and p38 MAPK, leading to SCF(Cdc4)-dependent ubiquitylation and proteasomal degradation of PGC-1alpha. Furthermore, SCF(Cdc4) negatively regulates PGC-1alpha-dependent transcription. We demonstrate that RNAi-mediated reduction of Cdc4 in primary neurons results in an increase of endogenous PGC-1alpha protein, while ectopic expression of Cdc4 leads to a reduction of endogenous PGC-1alpha protein. Finally, under conditions of oxidative stress in neurons, Cdc4 levels are decreased, leading to an increase in PGC-1alpha protein and PGC-1alpha-dependent transcription. These results suggest that attenuation of SCF(Cdc4)-dependent proteasomal degradation of PGC-1alpha has a role in mediating the PGC-1alpha-dependent transcriptional response to oxidative stress.

Neuronal activity and many pathological states in the CNS are accompanied by transient astrocytic swelling, which affects excitability, extrasynaptic transmission, and neuron-glia interactions. By using three-dimensional confocal morphometry (3DCM), we quantified the morphometric parameters of astrocytes in intact tissue. In experiments performed in brain cortex slices from transgenic GFAP/EGFP mice, we applied 3DCM to study the dynamic changes in astrocyte morphology during hypotonic stress. Our morphometric analysis showed that the effect of a 10-min application of hypotonic solution (200 mmol/kg) on the swelling of different cell compartments was dependent on the extent of the swelling of the total astrocyte volume. If the swelling of the whole cell, i.e., soma and processes, was less than approximately 10%, there were no differences between the swelling of the soma and the processes. However, if the swelling of the total cell volume was greater than 10%, the swelling of the processes was greater than the swelling of the soma. Analyzing the effect of hypotonic solution on the morphology of these astrocytes revealed that the total cell volume increased; however, certain cell compartments were distinguished in which the volume increased, whereas in other compartments cell volume decreased or apparently did not change, and the structure of some compartments was altered. Our data show that astrocytes in brain slices undergoing hypotonic stress display cell volume regulation as well as transient changes in morphology.

Survival of organisms requires the ability to adapt to changes in the environment. Adaptation of oxidative metabolism is essential for meeting increased energy demands in response to stressors, such as exposure to cold temperatures or increased physical activity. Adaptive changes in metabolism are often achieved at the level of gene expression, and nuclear receptors have prevalent roles in mediating such responses. Estrogen-related receptor alpha (ERRalpha) was the first orphan nuclear receptor to be identified, and yet its physiologic function remains unknown. Here, we show that mice lacking ERRalpha are unable to maintain body temperature when exposed to cold. Surprisingly, the inability to adapt to cold is not due to defects in the acute transcriptional induction of genes important for thermogenesis. Rather, we show that ERRalpha is needed for the high levels of mitochondrial biogenesis and oxidative capacity characteristic of brown adipose tissue (BAT), and thus for providing the energy necessary for thermogenesis. ERRalpha fulfills this role by acting directly at genes important for mitochondrial function, parallel to other factors controlling mitochondrial gene expression, such as NRF1 and NRF2/GABPA. Our findings demonstrate that ERRalpha is a key regulator of mitochondrial biogenesis and oxidative metabolism, and essential for adaptive thermogenesis.

Although research on improving child literacy is converging, no such body of research exists for adult literacy. Yet the need is no less significant. This study extends the knowledge garnered with younger populations by determining the reading comprehension strategies most important to adults' success on adult literacy outcome measures and aligning them with previously researched interventions. According to an analysis of key adult literacy outcome measures (i.e., competency-based, standardized tests of literacy commonly accepted as reasonable proxies for the global construct of adult literacy: Comprehensive Adult Student Assessment System [CASAS], General Educational Development [GED], and National Assessment of Educational Progress [NAEP]), adults should benefit from strategies that teach looking for clues in or generating questions about a text. Additionally, adults need to learn how to summarize and draw inferences in order to address higher-level literacy demands. Adult learners also need a metacognitive strategy to self-regulate reading behavior (e.g., choose a strategy to use, evaluate its effectiveness, and abandon and choose another strategy if necessary.) Furthermore, when using a competency-based standardized test, adult learners need to be coincidentally taught test-taking skills to reduce the test-related task demands and produce a better index of a learner's reading comprehension skills.

In the US health care system, a core safety net provider has two defining characteristics: (1) either by legal mandate or explicitly adopted mission, they maintain an "open door," offering patients services regardless of their ability to pay; and (2) a substantial portion of their patients are uninsured, on Medicaid, and/or otherwise vulnerable. The hospital Emergency Department (ED), by all accounts, falls within the definition of a core safety net provider. Yet many would argue that this is a primary health care role for which the ED was not originally intended or equipped. Should the ED be society's health-care safety net? Should it be the main provider of care for the indigent? Is this placing an unbearable strain on the ED? Should it be providing primary health-care? If not, what are the alternatives?

Two studies examined the influence of coping dispositions (repression, sensitization, and nondefensiveness) and anxiety on the encoding and memory representation of ambiguous threat-related stimuli. In Study 1, memory was tested shortly after encoding. Study 2 contrasted immediate and delayed testing. Repressers showed evidence of "mixed" affective reactions to ambiguous stimuli at encoding, accompanied by weak memory representation of potentially threatening implications of these stimuli. In contrast, sensitizers and anxious individuals manifested a processing bias in favor of threatening implications of ambiguous stimuli. Influences of coping on memory were most pronounced for delayed testing. Anxiety influences on memory were weak. An expectancy-based account of individual differences in processing ambiguous stimuli is discussed.

This article proposes the differentiation of Joy, Interest, and Activation in the Positive Affect (PA) scale of the Positive and Negative Affect Schedule (PANAS; D. Watson, L. A. Clark, & A. Tellegen, 1988). Study 1 analyzed the dynamic course of PA before, during, and after an exam and established the differentiation of the three facets. Study 2 used a multistate-multitrait analysis to confirm this structure. Studies 3-5 used success-failure experiences, speaking tasks, and feedback of exam results to further examine PA facets in affect-arousing settings. All studies provide convincing evidence for the benefit of differentiating three facets of PA in the PANAS: Joy, Interest, and Activation do have distinct and sometimes even opposite courses that make their separation meaningful and rewarding.

To clarify the impact of the classic gastrin/CCK-B receptor on the growth of benign and malignant colonic cells, two permanent cell lines expressing this receptor have been established. The conditionally immortalized nonmalignant colonic cell line YAMC and the colonic carcinoma cell line SW 403 were stably transfected with a plasmid encoding the gastrin/CCK-B receptor (GR), or with plasmid alone (V). Expression of the gastrin/CCK-B receptor in the transfected YAMC-GR and SW 403-GR cells was demonstrated by gastrin binding experiments. The YAMC-GR cell line did not respond mitogenically to pentagastrin or gastrin(17) in vitro and was not tumorigenic. The SW 403-GR cell line was stimulated by gastrin(17) in vitro, but the growth patterns of SW 403-GR and SW 403-V were the same in nude mice with cells injected either subcutaneously or into the spleen. These results provide further evidence that the gastrin/CCK-B receptor is not responsible for gastrin-stimulated growth in colonic tumors., (Copyright 1999 Academic Press.)

This study examined the influence of dispositional coping strategies (cognitive avoidance, vigilance) on priming and memory effects of emotional stimuli. In the first phase of the study participants performed a lexical decision task that involved threat-related and neutral words. Subsequently, a previously unannounced recognition memory test for a subset of the words presented during the first phase was carried out. Repressers (i.e. individuals high in avoidance and low in vigilance) showed stronger emotional priming effects than nonavoiders. Repressers also showed a memory deficit for emotional relative to neutral words, whereas sensitizers (vigilance high, avoidance low) remembered emotional words comparatively well. Results raise the question of whether repressers' memory deficits for threat-related stimuli are actually based on a less differentiated network of emotional information, as assumed by recent theoretical accounts of individual differences in coping.

The effect of nicotine on the release of noradrenaline and neuropeptide Y was investigated in isolated perfused guinea pig hearts (Langendorff technique). Endogenous noradrenaline, dihydroxyphenylglycol (both determined by high pressure liquid chromatography) and neuropeptide Y (determined by radioimmunoassay) were measured in the coronary venous effluent following the addition of nicotine to the perfusate. Nicotine (2 microM to 2 mM) dose-dependently increased both noradrenaline and neuropeptide Y overflow, and the release of both transmitters was closely correlated (r = 0.81). Despite ongoing nicotine administration noradrenaline and neuropeptide Y levels returned to basal values within 6 min of continuous nicotine administration indicating rapid tachyphylaxis to the effect of nicotine. The nicotine-induced release of noradrenaline and neuropeptide Y required the presence of extracellular calcium, and the release of both substances was suppressed by hexamethonium or by low concentrations of the inhibitors of the neuronal noradrenaline uptake (uptake1) desipramine and nisoxetine. Nicotine did not result in a significant release of the noradrenaline metabolite dihydroxyphenylglycol which was utilized as an indicator of free axoplasmic amine levels. The close correlation between noradrenaline and neuropeptide Y release, its calcium dependence, and the lack of dihydroxyphenylglycol overflow are in agreement with the concept of a common and exocytotic release of noradrenaline and neuropeptide Y induced by nicotine.

Behavioral momentum refers to the tendency for behavior to persist following a change in environmental conditions. The greater the rate of reinforcement, the greater the behavioral momentum. The intervention for noncompliance consisted of issuing a sequence of commands with which the subject was very likely to comply (i.e., high-probability commands) immediately prior to issuing a low-probability command. In each of five experiments, the high-probability command sequence resulted in a "momentum" of compliant responding that persisted when a low-probability request was issued. Results showed the antecedent high-probability command sequence increased compliance and decreased compliance latency and task duration. "Momentum-like" effects were shown to be distinct from experimenter attention and to depend on the contiguity between the high-probability command sequence and the low-probability command.

The overflow of neuropeptide Y (NPY; radioimmunoassay), noradrenaline and dihydroxyphenylethylenglycol (DOPEG; high pressure liquid chromatography) from guinea-pig perfused hearts was investigated in relationship to exocytotic and nonexocytotic release mechanisms. Exocytotic release: Electrical stimulation of the left stellate ganglion (12 Hz; 1 min) evoked a calcium-dependent overflow of noradrenaline and NPY, that was accompanied by a minor and prolonged increase in DOPEG overflow. This increase in DOPEG overflow was attenuated by blockade of neuronal amine re-uptake. In the presence of calcium, a closely related co-release of noradrenaline and NPY was also observed during administration of veratridine (10 microM); it was completely prevented by tetrodotoxin (1 microM). Nonexocytotic release: In the absence of extracellular calcium, veratridine (30 microM) induced noradrenaline overflow only when combined with the reserpine-like agent Ro 4-1284 (10 microM). This overflow was accompanied by efflux of DOPEG, but not of NPY. Similarily, tyramine (1-100 microM) induced a calcium-independent concomitant overflow of both noradrenaline and DOPEG, but not of NPY. During anoxic and glucose-free perfusion a predominantly calcium-independent overflow of noradrenaline was observed; only in the presence of extracellular calcium was this overflow accompanied by a minor overflow of NPY. Noradrenaline overflow, induced by veratridine plus Ro 4-1284 (in the absence of calcium), by tyramine, or by anoxia, was suppressed by blockade of neuronal amine re-uptake, and was, therefore, mediated by reversed transmembrane amine transport by the neuronal uptake carrier. The results indicate that NPY is co-released with noradrenaline only during calcium-dependent exocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)