Evidence had been provided that a disulfidelinked [12511iodotyramine/poly(D-lysine) conjugate was reductively cleaved when bound nonspecifically to the surface of Chinese hamster ovary (CHO) cells and that this cleavage was abolished by membrane-impermeant sulfhydryl blockers. The same blockers were subsequently found to inhibit the cytotoxicity of diphtheria toxin, a disulfide-linked heterodimer that binds to a specific surface receptor and must undergo chain separation to exert its cytotoxicity. This suggested that the disulfides of both macromolecules might be cleaved by a thioldisulfide interchange reaction, possibly mediated by protein disulfide-isomerase (PDI, EC 5.3.4.1). We tested whether inhibitors of PDI-in particular, bacitracin and anti-PDI antibodies-might mimic the two effects of sulfhydryl blockers. Both bacitracin and anti-PDI antibodies were effective in inhibiting both reductive processes. This strongly suggests that the disulfide cleavage in the two membrane-bound macromolecules is mediated by PDI and that this enzyme, besides its known retention in the endoplasmic reticulum, must also be exposed at the plasma membrane. This paper points to other potentially important disulfide reductions that might be catalyzed by surface-associated PDI. It thereby broadens the known functions of an enzyme already known for its multifunctional properties. (KDEL) sequence (3). It has also been reported to occur at the surface of mammalian cells (4, 5). This paper presents evidence that PDI is indeed responsible for cleaving the disulfides of these membrane-bound macromolecules and shows that cleavage is inhibited by monoclonal anti-PDI antibodies and by bacitracin, an antibiotic known to inhibit both the reductive (6) and the oxidative (7) functions of PDI.MATERIALS AND METHODS Materials. Bacitracin, DTNB, and pCMBS were purchased from Sigma. DT was from List Biological Laboratories, Campbell, CA. Thioredoxin and monobromotrimethylammoniobimane (Thiolyte MQ) were from Calbiochem. Lyphophilized ascites fluid containing anti-PDI monoclonal antibodies RL77, RL90, and HP13 (8) and the parent hybridoma cells were a gift from Charlotte S. Kaetzel, Case Western Reserve University School of Medicine. For some experiments the antibody-containing ascites fluid or conditioned medium was purified by protein G affinity chromatography (Mab Trap G kit from Pharmacia LKB). 125I-Tyn-SS-PDL conjugate was prepared as described (1). The experiments were carried out on Chinese hamster ovary (CHO) cells.Isolation of PDI and Assay of PDI and Thioredoxin. PDI was isolated from calf liver as described by Hillson et al. (9) and was 90-95% pure by electrophoresis. It was assayed as described by Carmichael et al. (10), except that the glutathione concentration was reduced to 250 AM. The amount and radioactivity of 125I-insulin were 50 ,ug and 105 cpm per sample. Under these conditions, PDI had a specific activity of 5200 units/mg in the standard 5-min assay. In the assays of Fig. 1 and Table 1, the reaction time was extended to 3...

Adverse posttraumatic neuropsychiatric sequelae (APNS) are common among civilian trauma survivors and military veterans. These APNS, as traditionally classified, include posttraumatic stress, post-concussion syndrome, depression, and regional or widespread pain. Traditional classifications have come to hamper scientific progress because they artificially fragment APNS into siloed, syndromic diagnoses unmoored to discrete components of brain functioning and studied in isolation. These limitations in classification and ontology slow the discovery of pathophysiologic mechanisms, biobehavioral markers, risk prediction tools, and preventive/ treatment interventions. Progress in overcoming these limitations has been challenging, because such progress would require studies that both evaluate a broad spectrum of posttraumatic sequelae (to overcome fragmentation) and also perform in-depth biobehavioral evaluation (to index sequelae to domains of brain function). This article summarizes the methods of the Advancing Understanding of RecOvery afteR traumA (AURORA) Study. AURORA conducts a large scale (n = 5,000 target sample) in-depth assessment of APNS development using a state-of-the-art battery of self-report, neurocognitive, physiologic, digital phenotyping, psychophysical, neuroimaging, and genomic assessments, beginning in the early aftermath of trauma and continuing for one year. The goals of AURORA are to achieve improved phenotypes, prediction tools, and understanding of molecular mechanisms to inform the future development and testing of preventive and treatment interventions.

Debate continues regarding the influence of litigation on pain outcomes after motor vehicle collision (MVC). In this study we enrolled European Americans presenting to the emergency department (ED) in the hours after MVC (n = 948). Six weeks later, participants were interviewed regarding pain symptoms and asked about their participation in MVC-related litigation. The incidence and predictors of neck pain and widespread pain six weeks after MVC were compared among those engaged in litigation ("litigants") and those not engaged in litigation ("non-litigants"). Among the 859/948 (91%) participants completing six week follow-up, 711/849 (83%) were non-litigants. Compared to non-litigants, litigants were less educated and had more severe neck pain, overall pain, and a greater extent of pain at the time of ED evaluation. Among individuals not engaged in litigation, persistent pain six weeks after MVC was common: 199/711 (28%) had moderate or severe neck pain, 92/711 (13%) had widespread pain, and 29/711 (4%) had fibromyalgia-like symptoms. Incidence of all three outcomes was significantly higher among litigants. Initial pain severity in the ED predicted pain outcomes among both litigants and non-litigants. Markers of socioeconomic disadvantage predicted worse pain outcomes in litigants but not non-litigants, and individual pain and psychological symptoms were less predictive of pain outcomes among those engaged in litigation. These data demonstrate that persistent pain after MVC is common among those not engaged in litigation, and provide evidence for bidirectional influences between pain outcomes and litigation after MVC.

Individual vulnerability factors influencing the function of the hypothalamic-pituitary-adrenal (HPA) axis may contribute to the risk of the development of persistent musculoskeletal pain after traumatic stress exposure. The objective of the study was to evaluate the association between polymorphisms in the gene encoding FK506 binding protein 51, FKBP5, a glucocorticoid receptor co-chaperone, and musculoskeletal pain severity six weeks after two common trauma exposures. The study included data from two prospective emergency department-based cohorts: a discovery cohort (n=949) of European Americans experiencing motor vehicle collision and a replication cohort of adult European American women experiencing sexual assault (n=53). DNA was collected from trauma survivors at the time of initial assessment. Overall pain and neck pain six weeks after trauma exposure were assessed using a 0–10 numeric rating scale. After adjustment for multiple comparisons, six FKBP5 polymorphisms showed significant association (minimum p <0.0001) with both overall and neck pain in the discovery cohort. The association of rs3800373, rs9380526, rs9394314, rs2817032, and rs2817040 with neck pain and/or overall pain six weeks after trauma was replicated in the sexual assault cohort, showing the same direction of the effect in each case. The results of this study indicate that genetic variants in FKBP5 influence the severity of musculoskeletal pain symptoms experienced during the weeks after motor vehicle collision and sexual assault. These results suggest that glucocorticoid pathways influence the development of persistent post-traumatic pain, and that such pathways may be a target of pharmacologic interventions aimed at improving recovery after trauma.