Pavlovian conditioning of opioid and nonopioid pain inhibitory mechanisms in humans.

Learning processes such as respondent or Pavlovian conditioning are believed to play an important role in the development of chronic pain, however, their influence on the inhibition of pain has so far not been assessed in humans. The purpose of this study was the demonstration of Pavlovian conditioning of stress-induced analgesia in humans and the determination of its opioid mediation. In a differential classical conditioning paradigm two different auditory stimuli served as conditioned stimuli and mental arithmetic plus white noise as unconditioned stimulus. Subsequent to four conditioning trials naloxone or placebo was applied in a double-blind fashion on two test days. Both pain threshold and pain tolerance showed conditioned stress-induced analgesia. Pain tolerance was affected by naloxone whereas pain threshold was not. The data of this study show that stress analgesia can be conditioned in humans and that it is at least partially mediated by the endogenous opioid system. Learning processes also influence pain inhibitory processes in humans and this effect might play a role in the development of chronic pain.     

Neuroelectric source imaging of steady-state movement-related cortical potentials in human upper extremity amputees with and without phantom limb pain

Whereas several studies reported a close relationship between changes in the somatotopic organization of primary somatosensory cortex and phantom limb pain, the relationship between alterations in the motor cortex and amputation-related phenomena has not yet been explored in detail. This study used steady-state movement-related cortical potentials (MRCPs) combined with neuroelectric source imaging to assess the relationship of changes in motor cortex and amputation-related phenomena such as painful and non-painful phantom and residual limb sensations, telescoping, and prosthesis use. Eight upper limb amputees were investigated. A significant positive relationship between reorganization of the motor cortex (distance of the MRCP source location from the mirrored source for hand movement) and phantom limb pain was found. Non-painful phantom sensations as well as painful and non-painful residual limb sensations were unrelated to motor cortical reorganization. A higher amount of motor reorganization was associated with less daily prosthesis use, which also tended to be related to more severe phantom limb pain. These results extend previous findings of a positive relationship between somatosensory reorganization and phantom limb pain to the motor domain and suggest a potential positive effect of prosthesis use on phantom limb pain and cortical reorganization.     

Glycan modulation and sulfoengineering of anti–HIV-1 monoclonal antibody PG9 in plants

Broadly neutralizing anti–HIV-1 monoclonal antibodies, such as PG9, and its derivative RSH hold great promise in AIDS therapy and prevention. An important feature related to the exceptional efficacy of PG9 and RSH is the presence of sulfated tyrosine residues in their antigen-binding regions. To maximize antibody functionalities, we have now produced glycan-optimized, fucose-free versions of PG9 and RSH in Nicotiana benthamiana. Both antibodies were efficiently sulfated in planta on coexpression of an engineered human tyrosylprotein sulfotransferase, resulting in antigen-binding and virus neutralization activities equivalent to PG9 synthesized by mammalian cells (^CHOPG9). Based on the controlled production of both sulfated and nonsulfated variants in plants, we could unequivocally prove that tyrosine sulfation is critical for the potency of PG9 and RSH. Moreover, the fucose-free antibodies generated in N. benthamiana are capable of inducing antibody-dependent cellular cytotoxicity, an activity not observed for ^CHOPG9. Thus, tailoring of the antigen-binding site combined with glycan modulation and sulfoengineering yielded plant-produced anti–HIV-1 antibodies with effector functions superior to PG9 made in CHO cells.Monoclonal antibodies (mAbs) offer great promise for AIDS treatment (1). In particular, the recent discovery of broadly neutralizing anti–HIV-1 mAbs (bNAbs) with extraordinary potency as exemplified by the antibodies PG9, PG16 (2), or those of the PGT series (3) creates hope for effective therapy by passive antibody transfer. PG9 and its close relative PG16 neutralize ∼80% of HIV-1 isolates across all clades (2, 4). The recognized epitopes are within the hypervariable and heavily glycosylated V1/V2 loops of the viral envelope glycoprotein gp120 and preferentially displayed in its trimeric state (2). Both mAbs use their unusually long complementarity-determining region (CDR) H3 domains (4–6) to penetrate the glycan shield of the virus and make contact with the underlying protein backbone (7). In addition, PG9 and PG16 recognize two highly conserved gp120 N-glycans attached to Asn¹⁶⁰ and Asn^156/173, which flank the peptide epitope (7–9). Remarkably, the glycan-binding properties of the two antibodies could be combined by modification of the PG9 light chain with R^L94SH^L95A as found in PG16. This PG9 variant (here termed RSH) has a superior neutralization capacity and broader coverage of HIV-1 isolates than either wild-type PG9 or PG16, which makes it an excellent choice for additional drug development studies (7).Proper N-glycosylation is important for aspects of mAb functionality, because the oligosaccharides attached to Asn²⁹⁷ of the crystallizable fragment (Fc) are known to strongly affect binding to cellular Fc receptors and thus, in vivo functionalities (10). In particular, core α1,6-fucosylation, typically found on mAbs produced in mammalian cell lines, has been shown to hinder antibody-dependent, cell-mediated cytotoxicity (ADCC) and antibody-dependent, cell-mediated virus inactivation (11), key effector functions in the context of anti–HIV-1 immune responses (12–14). Hence, considerable efforts have been undertaken to establish mAb production systems generating human-type N-glycans lacking this modification. Plants, particularly Nicotiana benthamiana, are well-suited for glycan engineering processes. The advantages of plant-based expression platforms include a high extent of glycan homogeneity, the flexibility with which glycosylation can be modulated, high production speed, and ease of large-scale production (15). The superior efficiency of glycocengineered mAbs produced in plants has recently been highlighted by ZMapp, an mAb mixture developed for the treatment of Ebola patients (16). Similarly, improved effector potency has been observed for plant-made anti–HIV-1 bNAb 2G12 (17), rendering glycoengineered plants an interesting production system for mAbs.Another posttranslational modification, namely tyrosine sulfation of selected residues in the CDR H3 region of PG9 and PG16, has recently been shown to be critical for high-affinity interactions with their antigen (4, 6). In humans, tyrosine sulfation is carried out by two closely related type II transmembrane proteins: tyrosylprotein sulfotransferase 1 (TPST1) and TPST2 (reviewed in refs. 18 and 19). Although plants contain TPSTs, these proteins are phylogenetically unrelated to the human enzymes and could, therefore, exhibit different enzymatic properties (18, 20). Previous attempts to produce bioactive PG9 and PG16 in N. benthamiana have failed, possibly because of deficient tyrosine sulfation (21). Hence, it is currently uncertain whether plant-based expression platforms are naturally capable of sulfating tyrosine residues in recombinant proteins.Here, we aimed to maximize the potency of bNAbs against HIV-1 using a plant-based expression system. For this goal, PG9 and RSH were expressed in a xylosyltransferase (XT)- and fucosyltransferase (FT)-deficient N. benthamiana mutant (ΔXT/FT) supporting the synthesis of glycan-optimized, fucose-free mAbs (15). Whereas tyrosine sulfation of PG9 by endogenous plant enzymes was barely detectable, this additional posttranslational modification was efficiently introduced by coexpression of human TPST1 (hsTPST1) modified with a plant Golgi-targeting sequence. When sulfated, plant-derived PG9 had essentially the same antigen-binding and virus neutralization properties as its counterpart produced in CHO cells. Importantly, ADCC activity was displayed by fucose-free, plant-produced mAbs but not by CHO-derived PG9. Furthermore, the controlled production of both sulfated and unmodified PG9 in the same expression system enabled us to establish the impact of tyrosine sulfation on the functionality of this important bNAb.     

Failure of extinction of fear responses in posttraumatic stress disorder: evidence from second-order conditioning

OBJECTIVES: Posttraumatic stress disorder (PTSD) is characterized by the re-experiencing of a traumatic event, although the trauma itself occurred in the past. The extinction of the traumatic response might be impeded if trauma reminders maintain fear responses by their association with the original trauma through second-order conditioning. METHOD: A differential conditioning paradigm with a trauma-specific picture, used as an acquired unconditioned stimulus, and graphic representations, used as conditioned stimuli, were employed in 14 PTSD patients, 15 trauma-exposed subjects without PTSD, and 15 healthy comparison subjects. The authors used event-related potentials of electroencephalogram (EEG), self-report measures, skin conductance responses, heart rate, and startle modulation to assess the differential conditioned response among subjects. RESULTS: Trauma-exposed subjects with and without PTSD but not healthy comparison subjects showed successful differential conditioning to the trauma-relevant cue indicative of second-order conditioning. Only PTSD patients exhibited enhanced conditioned responses to the trauma reminder during acquisition and impaired extinction as evident in more negative evaluations of the conditioned stimuli associated with a trauma reminder as well as enhanced peripheral and brain responses. CONCLUSIONS: These findings suggest that PTSD may be maintained by second-order conditioning where trauma-relevant cues come to serve as unconditioned stimuli, thus generalizing enhanced emotional responses to many previously neutral cues and impeding extinction. The extinction deficit in PTSD patients observed in this study underlines the need for therapies focusing on the extinction of learned responses, such as behavioral treatment, with or without the addition of pharmacological substances that enhance the extinction of a learned response.     

The impact of chronic pain in children and adolescents: Development and initial validation of a child and parent version of the Pain Experience Questionnaire

Psychosocial factors are crucial for understanding and treating chronic pain in adults, but also in children. For children, very few questionnaires for a multidimensional pain assessment exist. In adults, the Multidimensional Pain Inventory (MPI; [Kerns RD, Turk DC, Rudy TE. The West Haven-Yale Multidimensional Pain Inventory (WHYMPI). Pain 1985;23:345-56]) has been widely used to determine patients' adjustment to chronic pain. Using one section of the MPI as a model, we developed and evaluated the Pain Experience Questionnaire (PEQ) - child and parent version - that assesses the psychosocial impact of chronic pain in children and adolescents. As substantiated by confirmatory factor analysis in a sample of 111 children and adolescents (7-18 years) with chronic pain, the child PEQ entails the subscales pain severity, pain-related interference, affective distress and perceived social support. The parent version contains the subscales severity of the child's pain, interference and parental affective distress. Child and parent PEQ subscales were internally consistent. Age was unrelated to PEQ subscale scores. Girls and their mothers endorsed significantly greater pain severity, interference and affective distress. Validity analyses yielded a pattern of correlations with measures of depression, trait anxiety, pain activity, child behaviors, pain-related cognitions, and parenting behavior that is consistent with psychometric data of the adult MPI and previous findings on psychosocial aspects of chronic pediatric pain. Significant differences between children depending on patient status (participants in experimental or treatment studies, outpatients, inpatients) suggest external validity of the PEQ. Despite the preliminary nature of the psychometric evaluation, the child and parent PEQ seem promising for a comprehensive assessment of pediatric pain.     

Exposure to uncontrollable stress and the postimperative negative variation (PINV): prior control matters

The main goal of this study was to assess cortical functioning as indexed by the postimperative negative variation (PINV) induced by uncontrollable stress. Sixty-six persons were randomly assigned to three groups who underwent different sequences of stressor controllability. Within an S1-S2 paradigm, one group had initial control over aversive stimulation followed by loss of control and restitution of control. The other groups initially experienced either uncontrollability or controllability followed by a condition of control. Uncontrollable stress significantly enhanced PINV magnitudes independent of preceding control. However, control over aversive stimulation prior to loss of control normalized PINVs during restitution of control. Persons not experiencing prior control showed enhanced PINVs, longer reaction times, and more errors during restitution of control. We conclude that cortical activation changes are linked to the evaluation of instrumental contingencies. However, the exact determination of brain regions involved in the processing of uncontrollable stress needs further investigation.     

Effects of androgen deprivation on brain function in prostate cancer patients ¿ a prospective observational cohort analysis

ABSTRACT: BACKGROUND: Despite a lack of consensus regarding effectiveness, androgen deprivation therapy (ADT) is a common treatment for non-metastatic, low-risk prostate cancer. To examine a particular clinical concern regarding the possible impact of ADT on cognition, the current study combined neuropsychological testing with functional magnetic resonance imaging (fMRI) to assess both brain activation during cognitive performance as well as the integrity of brain connectivity. METHODS: In a prospective observational cohort analysis of men with non-metastatic prostate cancer at a Veterans Affairs medical center, patients receiving ADT were compared with patients not receiving ADT at baseline and at 6 months. Assessments included fMRI, the N-back task (for working memory), the stop-signal task (for cognitive control), and a quality of life questionnaire. RESULTS: Among 36 patients enrolled (18 in each group), 30 completed study evaluations (15 in each group); 5 withdrew participation and 1 died. Results for the N-back task, stop-signal task, and quality of life were similar at 6 months vs. baseline in each group. In contrast, statistically significant associations were found between ADT use (vs. non use) and decreased medial prefrontal cortical activation during cognitive control, as well as decreased connectivity between the medial prefrontal cortex and other regions involved with cognitive control. CONCLUSIONS: Although ADT for 6 months did not affect selected tests of cognitive function, brain activations during cognitive control and functional brain connectivity were impaired on fMRI. The long-term clinical implications of these changes are not known and warrant future study.     

Maladaptive plasticity, memory for pain and phantom limb pain: review and suggestions for new therapies

A number of studies have shown that phantom limb pain is associated with plastic changes along the neuraxis, with a close correlation between changes in the cortical representation of the affected limb and phantom limb pain. Mechanisms underlying these maladaptive plastic changes are related to a loss of GABAergic inhibition, glutamate-mediated long-term potentiation-like changes and structural alterations such as axonal sprouting. These plastic changes and phantom limb pain seem to be more extensive when chronic pain precedes the amputation. Behavioral interventions, stimulation, feedback and pharmacological interventions that are designed to reverse these maladaptive memory traces and enhance extinction may be beneficial for the treatment and prevention of phantom limb pain.     

Inhibitory Effects of Antagonists of Bombesin/Gastrin Releasing Peptide (GRP) and Somatostatin Analog (RC-160) on Growth of HT-29 Human Colon Cancers in Nude Mice

Nude mice bearing xenografts of HT-29 human colon cancer cell line were treated for 4 weeks with somatostatin analog (RC-160), bombesin/gastrin releasing peptide (GRP) antagonists (RC-3095 and RC-3440). In three separate experiments somatostatin analog RC-160 (50 μg/day) released from microgranules significantly reduced tumor growth. Bombesin/GRP antagonists, RC-3095 and RC-3440 injected subcutaneously (s.c.) twice daily at a dose of 10 μg had the greatest and consistently significant inhibitory effect on tumor growth. RC-3095 given once daily s.c. at a dose of 20 μg was less effective. RC-3095 also inhibited metastatic tumor growth after intrasplenic injection of HT-29 cells in nude mice. Specific binding sites of somatostatin, bombesin and epidermal growth factor (EGF) were detected on intact HT-29 cells or on the membranes from HT-29 tumor xenografts. The inhibitory effects of bombesin antagonists on tumor growth were consistently linked with a significant down-regulation of EGF receptors. Bombesin/GRP antagonists and somatostatin analogs could be considered for the development of new hormonal therapies for colon cancer.