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Background: Psoriasis is a chronic, inflammatory skin condition requiring long-term care. However, many psoriasis patients may not regularly receive care. Several factors affect access to care in the United States, including health insurance status. Additionally, it is unknown how health insurance status impacts the healthcare-seeking behavior of psoriasis patients. Healthcare-seeking behavior is broadly defined as an individual's actions to prevent or treat a perceived health problem, such as visiting a physician's office. Because early diagnosis and timely treatment improve patient outcomes, determining how insurance status impacts psoriasis patients' healthcare-seeking behavior and their ability to get care is important. This allows us to identify patients at risk for being untreated or undertreated. In this study, we aimed to assess the relationship between insurance status and (1) the degree to which psoriasis patients delay seeking or receiving care and (2) the degree to which psoriasis patients are unable to obtain care., Methods: This population-based study used 20 years of data from the Medical Expenditure Panel Survey from 2002 to 2021. We calculated descriptive statistics and performed adjusted multivariable logistic regression analyses., Results: We identified a weighted total of 4,506,850 psoriasis patients. Compared to those with private insurance, psoriasis patients with public-only insurance were 2.7 times more likely to delay seeking or receiving care (95% CI, 1.26-5.87). Compared to private insurance patients, uninsured psoriasis patients were 3.4 times more likely to be unable to obtain care (95% CI, 1.31-8.92). Compared to those with public-only insurance, uninsured psoriasis patients were 3.7 times more likely to be unable to obtain care (95% CI, 1.32-10.38)., Conclusions: This study found that psoriasis patients with public-only insurance were significantly more likely to delay seeking or receiving care compared to those with private insurance. This study also found that uninsured psoriasis patients were significantly more likely to be unable to obtain care than psoriasis patients with private insurance and those with public-only insurance. Developing strategies to increase healthcare access is necessary to ensure equitable, timely, and appropriate care for all psoriasis patients, regardless of their insurance status., Competing Interests: Declarations. Ethics approval and consent to participate: This study used publicly available, de-identified data; therefore, it was exempt from Institutional Review Board approval. Consent for publication: Not applicable. Competing interests: Authors KS, AH, RKY, LG, RK, HCT, SNB, and DKY declare that they have no competing interests. AWA has served as a research investigator, scientific advisor, or speaker to AbbVie, Amgen, Almirall, Arcutis, ASLAN, Beiersdorf, BI, BMS, EPI, Incyte, Leo, UCB, Janssen, Lilly, Mindera, Nimbus, Novartis, Ortho, Sun, Dermavant, Dermira, Sanofi, Takeda, Organon, Regeneron, Pfizer, and Ventyx. AbbVie, Amgen, Almirall, Arcutis, ASLAN, Beiersdorf, BI, BMS, EPI, Incyte, Leo, UCB, Janssen, Lilly, Mindera, Nimbus, Novartis, Ortho, Sun, Dermavant, Dermira, Sanofi, Takeda, Organon, Regeneron, Pfizer and Ventyx provided support in the form of salaries for AWA, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript., (© 2024. The Author(s).)

The transmembrane glycoprotein OX40 receptor (OX40) and its ligand, OX40L, are instrumental modulators of the adaptive immune response in humans. OX40 functions as a costimulatory molecule that promotes T cell activation, differentiation, and survival through ligation with OX40L. T cells play an integral role in the pathogenesis of several inflammatory skin conditions, including atopic dermatitis (AD). In particular, T helper 2 (T H 2) cells strongly contribute to AD pathogenesis via the production of cytokines associated with type 2 inflammation (e.g., IL-4, IL-5, IL-13, and IL-31) that lead to skin barrier dysfunction and pruritus. The OX40-OX40L interaction also promotes the activation and proliferation of other T helper cell populations (e.g., T H 1, T H 22, and T H 17), and AD patients have demonstrated higher levels of OX40 expression on peripheral blood mononuclear cells than healthy controls. As such, the OX40-OX40L pathway is a potential target for AD treatment. Novel therapies targeting the OX40 pathway are currently in development, several of which have demonstrated promising safety and efficacy results in patients with moderate-to-severe AD. Herein, we review the function of OX40 and the OX40-OX40L signaling pathway, their role in AD pathogenesis, and emerging therapies targeting OX40-OX40L that may offer insights into the future of AD management.

Background: This study investigated the accuracy in achieving proper lower limb alignment and component positions after total knee replacement (TKR) with image-free and image-based robotic-assisted TKR., Methods: A total of 129 patients (166 knees) suffering from end-stage knee arthritis who underwent TKA operated by robotic-assisted surgery between the years 2018 and mid-2021 were recruited. Radiological outcomes were compared between image-free and image-based robotic-assisted surgical systems., Results: There were significant differences between the two robotic systems when comparing the mean planned component alignment and the mean measured alignment on radiographs, in which the image-free robotic-assisted system was more varus, whereas the image-based robotic-assisted system was more valgus for both the mean femoral and tibial component coronal alignment (p < 0.001). For tibial component sagittal alignment, the image-based group had a larger deviation from the planned posterior slope (p < 0.001)., Conclusion: Image-free and image-based robotic assisted TKR had differing accuracy in femoral and tibial alignment., (© 2023 The Authors. The International Journal of Medical Robotics and Computer Assisted Surgery published by John Wiley & Sons Ltd.)

Introduction: Atypical femoral fractures (AFFs) are associated with delayed union and higher reoperation rates. Axial dynamization of intramedullary nails is hypothesized to reduce time-to-union (TTU) and fixation failure as compared to static locking., Methods: Consecutive acutely displaced AFFs fixed with long intramedullary nails across five centres between 2006 and 2021 with a minimum postoperative follow-up of three months were retrospectively reviewed. The primary outcome was TTU, compared between AFFs treated with dynamically or statically locked intramedullary nails. Fracture union was defined as a modified Radiographic Union Score for Tibial fractures score of 13 or greater. Secondary outcomes involved revision surgery and treatment failure, defined as non-union beyond 18 months or revision internal fixation for mechanical reasons., Results: A total of 236 AFFs (127 dynamically locked and 109 statically locked) were analysed with good interobserver reliability of fracture union assessment (intraclass correlation coefficient = 0.89; 95% CI = 0.82-0.98). AFFs treated with dynamized nails had significantly shorter median TTU (10.1 months; 95% CI = 9.24-10.96 vs 13.0 months; 95% CI = 10.60-15.40) (log-rank test, p = 0.019). Multivariate Cox regression revealed that dynamic locking was independently associated with greater likelihood of fracture union within 24 months (p = 0.009). Reoperations were less frequent in the dynamic locking group (18.9% vs 28.4%), although the difference was not statistically significant (p = 0.084). Static locking was an independent risk factor for reoperation (p = 0.049), as were varus reduction and lack of teriparatide use within three months of surgery. Static locking also demonstrated a higher frequency of treatment failure (39.4% vs 22.8%, p = 0.006) and was an independent predictor of treatment failure in logistic regression (p = 0.018). Other factors associated with treatment failure included varus reduction and open reduction., Conclusions: Dynamic locking of intramedullary nails in AFFs is associated with faster time to union, lower rate of non-union, and fewer treatment failures., Competing Interests: Declaration of Competing Interest Christian Fang is a shareholder of Lifespans Ltd and is also a speaker for Depuy Synthes. He has received research support from AO Foundation and Agnovos LLC. Frankie Leung is a shareholder of Lifespans Ltd and is also a speaker for Depuy Synthes, he has received research support from AO Trauma. All other authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

How Hispanic patients access dermatologic care for skin diseases is unknown. This study aims to determine if differences exist in accessing the emergency department (ED), primary care, and outpatient dermatologic offices for skin diseases between Hispanic and non-Hispanic White patients. This cross-sectional study used nationally representative data from the Medical Panel Expenditure Survey (MEPS) from 2016-2019. A total of 109,337,668 (weighted) patients with any skin disease diagnosed at an ED, primary care, or dermatology visit were identified. Hispanics comprised 13.0% and non-Hispanic Whites comprised 68.8% of this subpopulation. Overall, 94.1% of Hispanic patients attended a primary care visit for their skin complaint, 5.8% saw a dermatologist, and 0.1% attended an ED visit. Compared to non-Hispanic Whites, Hispanics were more likely to attend a primary care visit (aOR 1.865; 95%CI, 1.640-2.122) and less likely to attend an outpatient dermatology visit (aOR 0.536; 95%CI, 0.471-0.610), after adjusting for insurance status, education, income, sex, age, and comorbidities. Our study suggests that, compared to non-Hispanic Whites, Hispanic patients access primary care more frequently and outpatient dermatologic offices less frequently for their skin conditions. Language barriers, less familiarity with the healthcare system, and lack of adequate health insurance may play roles in this observation.

Competing Interests: Conflicts of interest: A.W.A. has served as a research investigator and/or scientific advisor to AbbVie, Almirall, Arcutis, ASLAN, Beiersdorf, BI, BMS, Dermavant, Dermira, EPI, Incyte, Janssen, Leo, Lilly, Modmed, Nimbus, Novartis, Ortho Dermatologics, Pfizer, Regeneron, Sanofi, Sun and UCB. C.Z-P., Sab.K., D.K.Y., Sam.K., N.M., R.R. and M.D.M. have no conflicts of interest to declare relevant to this manuscript.

Objectives: To determine the configuration of the distal fibula anatomy and the fitness of the Fibula Rod System (Acumed®, Hillsboro, Oregon) in a series of fibula models and to determine the optimal entry site of the rod., Methods: Consecutive series of computed tomography (CT) of tibias and fibulae with no fracture or deformity were converted to stereo-lithograph format, and imported into Meshmixer software (Autodesk, San Rafael, California). A 3.6 × 180 mm fibula rod model was virtually inserted to best fit the intramedullary canal of the fibula model and to a depth of 0 mm proud at the distal fibula. The location of the entry point in relationship to the fibular tip, and the distance between the rod and the lateral fibula cortex were measured., Results: CT of 41 fibulae (23 male and 18 female patients) contributed to the three-dimensional fibula modeling. The entry point was 3.5 mm (SD 2.0) medial to (in mortise view) and 1.0 mm (SD 2.1) anterior to (in lateral view) the fibular tip. The fibula rod was inserted to a depth of 6.2 mm (SD 2.1) proximal to the fibula tip. The mean shortest distance of the rod to the outer cortex was 1.88 mm (SD 0.87). There was a breach of the posterolateral cortex in one patient., Conclusion: The guide pin entry site of fibula rod should be medial and anterior offset with reference to the fibula tip, in contrary to the distal tip as recommended in the manual. There is a chance of breaching the posterolateral cortex with rod entry.

Introduction: Topical application of tranexamic acid (TXA) has been proposed as an alternative to intravenous administration to reduce perioperative bleeding in orthopaedic surgery. The purpose of this randomised controlled trial was to evaluate the efficacy and safety of 1 g topically applied TXA in patients undergoing fixation of intertrochanteric hip fractures by short femoral nailing., Methods: A total of 121 patients were enrolled between May 2018 and January 2020. Patients were randomly allocated (1:1) to receive either 10 mL (1 g) of TXA or 10 mL of normal saline (NS) injected through the subfascial drain following wound closure. Total blood loss, total drain output and blood transfusion requirements up to postoperative day 3 were recorded. Rates of thromboembolic complications and mortality up to 90 days postoperatively were also compared., Results: There was no statistically significant difference in total blood loss, total drain output or proportion of patients requiring transfusions. Median total blood loss was 1.088 L (IQR: 0.760-1.795) in the TXA group and 1.078 L (IQR: 0.797-1.722) in the NS group (P = .703). Median total drain output was 60 mL (IQR: 40-140) in the TXA group and 70 mL (IQR: 30-168) in the NS group (P = .696). Blood transfusions were administered in 29 patients (47.5%) in the TXA group and 27 patients (45.0%) in the NS group (P = .782). There was also no difference in frequency of thrombotic complications or mortality within 90 days. There were five thrombotic events in the TXA group and four in the NS group (P = .751). The 90-day mortality rate was 6.6% (4 patients) in the TXA group and 3.3% (2 patients) in the NS group (P = .680)., Conclusion: A 1 g dose of topically administered TXA did not produce any difference in blood loss, transfusion requirements, thromboembolic complications, or 90-day mortality. Future trials may consider the effect of larger doses in patients undergoing hip fracture fixation surgery., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Percutaneous insertion of third-generation straight humeral nails is a recent alternative to the conventional open method. Rather than splitting, retracting and subsequently repairing the supraspinatus fibers to visualize the humeral head entry site, the percutaneous approach utilizes a cannulated awl to enter the intramedullary canal through the supraspinatus fibers without visualizing internal shoulder structures. Despite recent evidence demonstrating satisfactory outcomes in the percutaneous method, the potential for iatrogenic injury to the rotator cuff and other shoulder structures is not fully understood., Materials and Methods: We performed an anatomical study of 46 shoulders in 23 cadavers to compare damage caused to internal shoulder structures between the open and percutaneous techniques. Dimensions and morphologies of supraspinatus and humeral head perforations were recorded., Results: The percutaneous technique produced greater latitudinal tearing ( p = 0.002) and less longitudinal tearing ( p < 0.001) of muscle fibers, however there was no difference in supraspinatus hole area ( p = 0.748). The long head biceps tendon was within 3 mm of the bone entry hole in 13 (28%) shoulders, with one shoulder in the open group exhibiting full tendon transection., Conclusions: Percutaneous insertion of intramedullary nails using a cannulated awl appears to produce similar soft tissue and bone entry site morphology as compared to the conventional open technique. The percutaneous method was associated with slightly greater latitudinal tearing, however the effects of this remain to be clarified through clinical studies. External rotation should be avoided during instrumentation to reduce the risk of biceps tendon transection.

Introduction: The relationship between patient expectations and clinical outcomes has recently been of increasing interest in the field of orthopaedics. The objective of this study was to evaluate the relationships between (1) patient pre-treatment expectations and post-treatment clinical outcomes, and (2) fulfillment of expectations and patient satisfaction, following distal radius fracture., Methods: This was a prospective multicenter cohort study conducted across three hospitals. A total of 133 patients admitted to hospital between 2016 and 2018 with a distal radius fracture were recruited. Patients were administered the Trauma Expectation Factor (TEF) at baseline to measure expectations of pain and functioning at one-year post-injury. Patients were followed up at 6 and 12 months post-injury and outcomes were measured using the Trauma Outcome Measure (TOM), Shortened Disabilities of the Arm, Shoulder and Hand (QuickDASH) questionnaire, and Short-Form 12-item (SF-12) health survey. Fulfillment of expectations was measured as TEF score minus TOM score at each time point. Patient satisfaction was also measured using the Patient Satisfaction Questionnaire Short Form (PSQ-18). Multivariate regression models were created to evaluate the effect of expectations on outcomes., Results: Patient expectations correlated moderately with outcomes at 6 and 12 months post-injury. In the multivariate analysis, patient expectations were predictive of better outcomes at 6 and 12 months. Injury severity, age, mechanism of injury, and whether the injury occurred on duty also contributed significantly to one or more regression models. Patient satisfaction correlated weakly with fulfillment of expectations at 6 months, but moderately with all outcome scores. At 12 months, satisfaction did not correlate significantly with expectations fulfillment but correlated moderately to highly with all outcome scores., Conclusion: Patient expectations independently predicted outcomes at 6 and 12 months post-injury. Standardized assessment and management of patient expectations may be relevant to future clinical practice and research to best quantify and optimize patient outcomes., Competing Interests: Declarations of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Purpose: Many standardized outcome measures exist to measure recovery after surgical fixation of distal radius fractures, however, choosing the optimal instrument is difficult. We evaluated responsiveness, ceiling/floor effects, and criterion validity over multiple time intervals across a 2-year follow-up period for six commonly used instruments., Methods: A total of 259 patients who received open reduction and internal fixation for distal radius fractures between 2012 and 2015 were recruited. Patients were administered the Patient-Rated Wrist Evaluation ( PRWE ), Shortened Disabilities of the Arm, Shoulder and Hand questionnaire ( QuickDASH ), Green and O'Brien score (Cooney modification) ( CGNO ), Gartland and Werley score (Sarmiento modification) ( SGNW ), flexion-extension arc ( FEArc ), and grip fraction test ( GripFrac ) at 1.5, 3, 6, 12, and 24 months postoperatively. Responsiveness was evaluated by calculating standardized response means (SRM) and Cohen's d effect sizes (ES), and by correlating each instrument's change scores against those of QuickDASH and PRWE , which were also used as external comparators to assess criterion validity. Ceiling/floor effects were calculated for all measures at each time point., Results: SRM (1.5-24 months) were 1.81, 1.77, 1.43, 1.16, 2.23, 2.45 and ES (1.5-24 months) were 1.81, 1.82, 1.95, 1.31, 1.99 and 2.90 for QuickDASH, PRWE, CGNO, SGNW, FEArc, and GripFrac respectively. Spearman correlation coefficients against QuickDASH at 24 months were: 0.809, 0.248, 0.563, 0.285, and 0.318 for PRWE, CGNO, SGNW, FEArc, and GripFrac respectively. Significant (>15% of patients reaching maximum score) ceiling effects were observed before 6 months for PRWE and SGNW ., Conclusions: Our evidence supports the use of QuickDASH , PRWE , FEArc and GripFrac up to 6 months postsurgery, and QuickDASH and PRWE after 6 months., Level of Evidence: Level II.

Introduction: Patella fractures managed by fixation with metal implants often cause local soft tissue irritation and necessitate implant removal. An alternative is to utilize suture-based fixation methods. We have adopted suture and hybrid fixation in the routine management of patella fractures. Here, we compare the results of 3 fixation techniques., Materials and Methods: Eighty-seven eligible patients underwent patella fracture fixation over a 3-year period. As determined by fracture configuration, patients received (1) suture fixation (transosseous sutures and figure-of-eight tension banding with FiberWire), (2) hybrid fixation (transosseous FiberWire sutures and metal tension banding), or (3) metal fixation. Primary outcome measures included reoperation rate and soft tissue irritation. Secondary outcomes included surgical complications, radiological, and functional parameters., Results: Reoperation rate was highest for metal fixation (25/57, 43.9%) and lowest for suture fixation (2/13, 15.4%). Soft tissue irritation necessitating implant removal was the predominant reason for reoperation and was significantly less prevalent following suture fixation (1/13, 7.7%, P < .01). Hybrid fixation resulted in similar rates of soft tissue irritation (6/17, 35.3%) and implant removal (7/17, 41.2%) as compared to metal fixation. There was a significant increase in patella baja (13/17, 76.5%) and reduction in Insall-Salvati ratio (0.742; 95% confidence interval: 0.682-0.802) following hybrid fixation as compared to the other 2 fixation methods ( P < .05)., Discussion: Suture fixation results in the least amount of soft tissue irritation and lowest reoperation rate, but these advantages are negated with the addition of a metal tension band wire. Hybrid fixation also unbalances the extensor mechanism., Conclusion: Patients should be counseled as to the expected sequelae of their fixation method. Suture fixation is the favored means to fix distal pole fractures of the patella. An additional metal tension band loop may confer additional stability but should be applied with caution., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objectives: This study aims to identify if wintertime surgery increases the mortality of the patients after hip fracture operations., Design: Retrospective observational cohort study., Setting: The data for this citywide retrospective observational cohort study came from Clinical Data Analysis Reporting System., Patient: This study included 35 409 patients with hip fracture operations from July 2005 to December 2013., Main Outcome Measures: Cox regression hazard model was used to estimate the independent effect of operation being performed in winter on the hazard of mortality. The hazard model included covariates found to be independent predictors of mortality: age, sex, surgical delay, and Charlson Comorbidity Index (CCI)., Results: There was a seasonal variation with more hip fracture operations happening in the winter months. The 1-month, 6-month, 1-year, and 5-year mortality were 3%, 11%, 17%, and 47%, respectively. Operation performed in winter was associated with a higher hazard of mortality (hazard ratio [HR] 1.040; 95% confidence interval: 1.010-1.072; P = .009). The HR was greater with male sex (HR 1.786; P = .000), advanced age (≥85 years old: HR 2.819; P = .000), the longer surgical delay (HR 1.018; P = .000), and higher CCI (severe CCI group: HR 2.963; P = .000)., Conclusion: Wintertime hip fracture surgery was associated with an increased hazard of mortality after adjusting for other known risk factors affecting mortality post hip fracture operations., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: To develop a reference for the distal screw length in volar locking plate fixation for distal radial fractures in an East Asian population., Methods: 12 pairs of forearm specimens from 11 male and one female East Asian cadavers were scanned using computed tomography. On sagittal images of the distal radius, the mean cortex-to-cortex distance of 8 quadrants was measured as a reference for the distal screw length. In addition, intra-operative 3-dimensional fluoroscopy of 10 male and 10 female patients who underwent volar locking plate fixation for distal radial fractures was used to validate the distal screw length in the cadaveric reference. 76 distal locking screws were applied in the 8 quadrants; their cortex-to-cortex distances were measured., Results: The mean cortex-to-cortex distances at quadrants A, B, C, D, E, F, G, and H were 15.4 mm, 19.6 mm, 20.8 mm, 20.0 mm, 13.3 mm, 18.0 mm, 18.8 mm, and 17.4 mm, respectively. In 45% of the specimens, the 2 screws inserted at quadrants C and D were longest. Distal screws (quadrants A to D) were significantly longer than proximal screws (quadrants E to H) [p=0.02]. In intra-operative 3-dimensional fluoroscopic images, 2 of the 76 distal locking screws penetrated the dorsal cortex (one in quadrant A and one in quadrant F). The mean screw length was 88.0% of the cortex-to-cortex distance. Referenced to the cadaveric data, 88.2% of the screws could be safely inserted without penetrating the dorsal cortex, and the remaining 11.8% of screws (5 at quadrant D, one at quadrant B, and 3 at quadrant C; all in female patients) could potentially cause dorsal cortex penetration of 2 to 4 mm. In male patients, the mean screw length was 76.1% of the cortex-to-cortex distance based on the cadaveric reference. In female patients, when the screw length was 4 mm less than the cadaveric reference, the mean screw length would be 72.0% of the referenced cortex-to-cortex distance, with no dorsal cortex penetration., Conclusion: In female patients, the screw length should be 4 mm less than the cadaveric reference to avoid dorsal cortex penetration.

Angiotensin II (AngII) and aldosterone cooperate centrally to produce a robust sodium appetite. The intracellular signaling and circuitry that underlie this interaction remain unspecified. Male rats pretreated with both deoxycorticosterone (DOC; a synthetic precursor of aldosterone) and central AngII exhibited a marked sodium intake, as classically described. Disruption of inositol trisphosphate signaling, but not extracellular-regulated receptor kinase 1 and 2 signaling, prevented the cooperativity of DOC and AngII on sodium intake. The pattern of expression of the immediate early gene product cFos was used to identify key brain regions that may underlie this behavior. In the paraventricular nuclei (PVN) of the hypothalamus, DOC pretreatment diminished both AngII-induced cFos induction and neurosecretion of oxytocin, a peptide expressed in the PVN. Conversely, in the organum vasculosum lateral terminalis (OVLT), DOC pretreatment augmented cFos expression. Immunohistochemistry identified a substantial presence of oxytocin fibers in the OVLT. In addition, when action potentials in the PVN were inhibited with intraparenchymal lidocaine, AngII-induced sodium ingestion was exaggerated. Intriguingly, this treatment also increased the number of neurons in the OVLT expressing AngII-induced cFos. Collectively, these results suggest that the behavioral cooperativity between DOC and AngII involves the alleviation of an inhibitory oxytocin signal, possibly relayed directly from the PVN to the OVLT., (Copyright © 2014 the authors 0270-6474/14/349249-12$15.00/0.)

Erythropoietin (EPO) is the primary regulator of red blood cell development. Although hypoxic regulation of EPO has been extensively studied, the mechanism(s) for basal regulation of EPO are not well understood. In vivo studies in healthy human volunteers and animal models indicated that angiotensin II (Ang II) and angiotensin converting enzyme inhibitors regulated blood EPO levels. In the current study, we found that Ang II induced EPO expression in situ in murine kidney slices and in 786-O kidney cells in culture as determined by reverse transcription polymerase chain reaction. We further investigated the signaling mechanism of Ang II regulation of EPO in 786-O cells. Pharmacological inhibitors of Ang II type 1 receptor (AT1R) and extracellular signal-regulated kinase 1/2 (ERK1/2) suppressed Ang II transcriptional activation of EPO. Inhibitors of AT2R or Src homology 2 domain-containing tyrosine phosphatase had no effect. Coimmunoprecipiation experiments demonstrated that p21Ras was constitutively bound to the AT1R; this association was increased by Ang II but was reduced by the AT1R inhibitor telmisartan. Transmembrane domain (TM) 2 of AT1R is important for G protein-dependent ERK1/2 activation, and mutant D74E in TM2 blocked Ang II activation of ERK1/2. Ang II signaling induced the nuclear translocation of the Egr-1 transcription factor, and overexpression of dominant-negative Egr-1 blocked EPO promoter activation by Ang II. These data identify a novel pathway for basal regulation of EPO via AT1R-mediated Egr-1 activation by p21Ras-mitogen-activated protein kinase/ERK kinase-ERK1/2. Our current data suggest that Ang II, in addition to regulating blood volume and pressure, may be a master regulator of erythropoiesis.

Joint aspiration is a reliable tool for diagnosis of periprosthetic infection. There are different indications, techniques, and approaches for joint aspiration. We recommend that it be performed selectively when infection is suspected clinically. The specimens should be interpreted based on the results of the culture as well as the white cell count and differential. Specimen collection, transport, and analysis should be prompt to ensure yield accuracy.

Purpose: The purpose of this study was to examine the difference in the position of bone tunnels prepared by the transportal technique versus the transtibial technique in anterior cruciate ligament (ACL) reconstruction., Methods: A consecutive series of 42 patients receiving single-bundle ACL reconstructions were recruited between July 1, 2007, and December 31, 2008. The preparations of the femoral tunnel were performed by the transtibial technique in the first 21 cases and by the transportal technique in the subsequent 21 cases. Magnetic resonance imaging examination was performed in 39 patients (93%) 1 year after the index operation (20 transtibial and 19 transportal). Optimal tunnel position was defined as a lateralized femoral tunnel at a position of less than 11 o'clock for a right knee or more than 1 o'clock for a left knee, an adequate posteriorized femoral tunnel in the fourth quadrant of the modified Bernard line, and a tibial tunnel located in the second quadrant of the modified Amis line., Results: The average clock position was 10:18 in the transportal group and 10:54 in the transtibial group (P < .001). Five outliers were found in the transtibial group but none in the transportal group. Concerning the femoral tunnel position on the sagittal-cut magnetic resonance imaging scan, the average position along the modified Bernard line was 74% in the transportal group and 69% in the transtibial group (P = .029). Concerning the tibial tunnel position, the average tibial tunnel positions along the modified Amis line were 47% and 52%, respectively (P = .019)., Conclusions: The adoption of the transportal technique in single-bundle ACL reconstruction produced improved positions in both the femoral and tibial tunnels when compared with the transtibial technique., Level of Evidence: Level III, case-control study., (Copyright © 2013 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

The renin-angiotensin-aldosterone system makes a critical contribution to body fluid homeostasis, and abnormalities in this endocrine system have been implicated in certain forms of hypertension. The peptide hormone angiotensin II (AngII) regulates hydromineral homeostasis and blood pressure by acting on both peripheral and brain targets. In the brain, AngII binds to the angiotensin type 1 receptor (AT1R) to stimulate thirst, sodium appetite and both arginine vasopressin (AVP) and oxytocin (OT) secretion. The present study used an experimental model of endogenous AngII to examine the role of p44/42 mitogen-activated protein kinase (MAPK) as a signalling mechanism to mediate these responses. Animals were given a combined treatment of furosemide and a low dose of captopril (furo/cap), comprising a diuretic and an angiotensin-converting enzyme inhibitor, respectively, to elevate endogenous AngII levels in the brain. Furo/cap induced p44/42 MAPK activation in key brain areas that express AT1R, and this effect was reduced with either a centrally administered AT1R antagonist (irbesartan) or a p44/42 MAPK inhibitor (U0126). Additionally, furo/cap treatment elicited water and sodium intake, and irbesartan markedly reduced both of these behaviours. Central injection of U0126 markedly attenuated furo/cap-induced sodium intake but not water intake. Furthermore, p44/42 MAPK signalling was not necessary for either furo/cap- or exogenous AngII-induced AVP or OT release. Taken together, these results indicate that p44/42 MAPK is required for AngII-induced sodium appetite but not thirst or neurohypophysial secretion. This result may allow for the discovery of more specific downstream targets of p44/42 MAPK to curb sodium appetite, known to exacerbate hypertension, at the same time as leaving thirst and neurohypophysial hormone secretion undisturbed., (© 2012 British Society for Neuroendocrinology.)

Study Design: A retrospective review of infective spondylitis patients assessed at a major, tertiary referral centre in Hong Kong., Objective: To assess the prevalence, risk factors, clinical features, and prognostic outcomes associated with tuberculous spondylitis to that of pyogenic spondylitis in Southern Chinese treated at a single institution., Summary of Background Data: Previous studies in Asia suggest that tuberculous spondylitis is the predominant infection unless proven otherwise. Current clinical experiences suggest otherwise; however, the current trend and clinical profile of infective spondylitis among Southern Chinese remains speculative with no published studies examining their prevalence., Methods: A retrospective review was performed of all infective spondylitis cases presenting from January 2004 to July 2008 to a tertiary referral center. Cases were included on the basis of clinical and microbiological criteria. Radiographic imaging was used for further confirmation., Results: Ninety-one patients were identified. Overall, tuberculous spondylitis and pyogenic spondylitis entailed 22 (24.2%) and 69 (75.8%) cases, respectively. Staphylococcus aureus was the most commonly isolated infective agent associated with pyogenic spondylitis. Individuals with pyogenic spondylitis were significantly much older than those with tuberculous spondylitis (P = 0.001). Intravenous drug addiction was the most commonly noted risk factor followed by diabetes, and found to be more prevalent in pyogenic spondylitis cases. At initial presentation, white cell count and c-reactive protein levels were higher in pyogenic spondylitis cases compared with tuberculous spondylitis (P < 0.05). The occurrence of tuberculous spondylitis cases was predominant in the thoracic region (40.9%) (P < 0.05). Surgical intervention was performed in 54.5% of tuberculous spondylitis and in 24.6% of the pyogenic spondylitis cases (P = 0.009)., Conclusion: In Southern Chinese, compared to previous reports over the past 3 decades, a changing prevalence of decreasing tuberculous spondylitis was observed. Pyogenic spondylitis was found to be more common among patients hospitalized for infective spondylitis. This has important implications on the method of diagnosis and the need for establishing microbiological diagnosis before commencing treatment. "Best guess" therapy should not be targeted at tuberculous spondylitis only. There are clear distinctions in the biologic and clinical profiles between tuberculous and pyogenic spondylitis that would help to direct therapy.

In addition to its well known stimulation of cAMP production, the human melanocortin type 4 (hMC4) receptor recently has been shown to mediate p44/42 MAPK activation. This finding opens new questions about the structural and signaling mechanisms that connect the receptor to this alternate cell signaling pathway. Point mutants in the hMC4 receptor that have been associated with obesity were constructed and transfected into HEK 293 cells. Functional analyses then were done to determine if these mutations would similarly impact cAMP formation and p44/42 MAPK signaling. Whereas a D90N mutation in the second transmembrane domain and a D298A mutation in the seventh transmembrane domain impaired both cAMP formation and p44/42 MAPK activation, a more conservative D298N mutation retained cAMP formation but abolished p44/42 MAPK activation. The D298N mutation identified, for the first time, differential structural requirements of the hMC4 receptor for activation of the cAMP and p44/42 MAPK pathways. Furthermore, functional characterizations of a series of chimeric receptors combining the hMC4 receptor and the hMC3 subtype, a receptor that does not couple to p44/42 MAPK activation despite stimulating adenylyl cyclase, indicate that the hMC4 cytoplasmic tail is a necessary structural element for p44/42 MAPK signaling. Subsequent investigation of the signaling requirements for p44/42 MAPK activation demonstrated that the adenylyl cyclase inhibitor 2', 5'-dideoxyadenosine blocked agonist-induced p44/42 MAPK activation, but the PKA inhibitor Rp cAMPS did not. Taken together, these data indicate that cAMP is required, but not sufficient for p44/42 MAPK activation and suggest structural elements required for hMC4 receptor signaling.

Angiotensin II plays a key role in the regulation of body fluid homeostasis. To correct body fluid deficits that occur during hypovolaemia, an animal needs to ingest both water and electrolytes. Thus, it is not surprising that angiotensin II, which is synthesized in response to hypovolaemia, acts centrally to increase both water and NaCl intake. Here, we review findings relating to the properties of angiotensin II receptors that give rise to changes in behaviour. Data are described to suggest that divergent signal transduction pathways are responsible for separable behavioural responses to angiotensin II, and a hypothesis is proposed to explain how this divergence may map onto neural circuits in the brain.

Purpose: To evaluate the relationship between retinal nerve fiber layer (RNFL) thickness measured by optical coherence tomography (OCT) and the axial length/refractive error of the eye., Methods: A total of 115 eyes of 115 healthy subjects, comprising 75 eyes with high myopia (spherical equivalent [SE] < -6.0 D) and 40 eyes with low to moderate myopia (SE between -6.0 D and -0.5D), were analyzed in this cross-sectional study. Total average and mean clock hour RNFL thicknesses were measured by OCT and compared between the two myopia groups. Associations between RNFL measurements and axial length and spherical equivalent were evaluated by linear regression analysis., Results: The RNFL measurements were significantly lower in the high myopia group compared with those of the low-to-moderate myopia group at 12, 1, and 7 o'clock (right eye orientation). Apart from the temporal clock hours, significant correlations were evident between RNFL measurements and the axial length and spherical equivalent. The average RNFL thickness decreased with increasing axial length (r = -0.314, P = 0.001) and negative refractive power (r = 0.291, P = 0.002). A significant proportion of myopic eyes were classified as outside normal limits, with reference to the normative database. The most frequently abnormal sector was at 2 o'clock, where 16.5% of myopic eyes were outside normal limits., Conclusions: RNFL measurements vary with the axial length/refractive error of the eye. Analysis of RNFL thickness in the evaluation of glaucoma should always be interpreted with reference to the refractive status. Although the normative database provided by OCT has been helpful in identifying ocular diseases involving the RNFL, it may not be reliable in the analysis of myopic eyes.

Although the intrareceptor mechanisms whereby the angiotensin II (AngII) type 1 receptor activates phospholipase C (PLC) have been extensively investigated, analogous studies of signaling through mitogen-activated protein kinases (MAPK) have been lacking. We investigated MAPK activation and traditional G(q)/PLC signaling in transfected cells using AngII and the signaling selective agonist [Sar(1),Ile(4),Ile(8)] AngII (SII). SII stimulated MAPK without inositol trisphosphate (IP(3)) production and thereby stabilizes an activated receptor state linked to G protein-independent MAPK signaling. Using receptor mutagenesis, we focused on the seventh transmembrane domain and identified three key residues-Tyr(292), Phe(293), and Thr(287). At least three distinct activated states were revealed: 1) an AngII-stabilized state linked to G(q)/PLC signaling, 2) an AngII-stabilized state connected to G protein-independent MAPK activation, and 3) a SII-stabilized state associated with G protein-independent MAPK signaling. The mutant Y292F failed to exhibit AngII-induced IP(3) turnover yet remained capable of AngII-induced MAPK activation. SII failed to stimulate MAPK in Y292F-transfected cells. Thus, Tyr(292) is a key epitope for activated states 1 and 3 but not required for activated state 2. Although the F293L mutant retained normal AngII responses, it also showed an IP(3) response to SII, indicating that Phe(293) may be involved in constraining the receptor to its inactive state. Mutations of Thr(287) abolished all SII-induced signaling without affecting any AngII responses. Thr(287) therefore represents a key residue for a SII-stabilized activated state. Taken together, the data identified a novel structural requirement (Thr(287)) for the SII-stabilized activated state and redefined the mechanistic roles for Tyr(292) and Phe(293).

Central injections of angiotensin II (AngII) increase both water and NaCl intake. These effects of AngII occur largely through stimulation of the AngII type 1 (AT(1)) receptor. Stimulation of the AT(1) receptor leads to a number of intracellular events, including phospholipase C (PLC) activation and the subsequent formation of diacylglycerol and inositol trisphosphate (IP(3)), which then activate protein kinase C (PKC) and increase intracellular calcium, respectively. In addition, AT(1) receptor stimulation leads to the activation of MAPK family members. Recent experiments using mutated AT(1) receptor constructs or the AngII analog Sar(1),Ile(4),Ile(8)-AngII (SII) revealed that MAPK activation can occur independent of PLC/PKC/IP(3) activation. The present experiments used in vitro and in vivo approaches to clarify the cellular and behavioral responses to SII. Specifically, SII mimicked AngII stimulation of MAPK in AT(1) receptor-transfected COS-1 cells and rat brain but blocked the effects of AngII in two distinct settings: in vitro stimulation of IP(3) and in vivo increases in water intake. Moreover, SII increased intake of 1.5% NaCl, despite the SII blockade of IP(3) formation and water intake. Examination of brain tissue showed increases in Fos expression in several AngII-sensitive brain areas after injection of AngII, but not SII. The lack of SII-induced IP(3) production, water intake, and Fos expression strongly suggest that the PLC/PKC/IP(3) pathway is required for water intake, but not NaCl consumption stimulated by AngII. Collectively, these results support the hypothesis that divergent intracellular signals from a single receptor type can give rise to separable behavioral phenomena.

Examination of the Rattus norvegicus genome reveals differences in the melanocortin 3 receptor (MC3R) compared with the published sequence (accession X70667). To clarify these differences, we used RT-PCR to clone MC3R from Sprague Dawley rats. These efforts revealed a sequence for the rat MC3R consistent with that predicted by the rat genome, but different from the published receptor by three amino acids, all of which were located in the predicted second transmembrane domain (TM2). Analysis of these residues revealed that TM2 of the rat MC3R is more homologous with other species than previously considered. The presently described sequence maps onto chromosome 3 of the rat genome, which shows highly conserved synteny with the mouse chromosome 2 and the human chromosome 20. Transient expression revealed high affinity binding of [125I]-NDP-MSH and a concentration-dependent cAMP response to the synthetic agonist MTII. These data both clarify the sequence of the MC3R and demonstrate the great utility of genomic information recently made available.

The central melanocortin system has emerged as a potential regulator of food intake. This action of melanocortins appears to occur through intrahypothalamic, melanocortin-containing projections, including those from the arcuate to the paraventricular nucleus (PVN). Although the complexity of feeding behavior and the long duration of the effects of melanocortins on food intake suggest changes in gene expression, the mechanism by which such changes occur has been elusive. In the present report, we describe experiments using in vitro and in vivo approaches to demonstrate melanocortin-induced phosphorylation (activation) of members of the mitogen-activated protein kinase (MAPK) family of transcription factors. First, application of the melanocortin agonist MTII to COS-1 cells resulted in an increase in phosphorylated MAPK after the cells were transfected with the melanocortin type 4 receptor (MC4-R), but not the type 3 receptor. Formation of cAMP, however, was observed when either receptor subtype was transfected. Subsequent experiments revealed that the effect of MTII on MAPK activation in MC4-R-transfected cells was dose-dependent and was maximal after 10 min of MTII exposure. Second, central injections of MTII increased the number of phospho-MAPK-immunoreactive cells in the rat PVN compared to vehicle-injected animals. When coupled with immunohistochemical identification of PVN neurons containing oxytocin, a clear segregation was apparent, allowing for a precise anatomical description of the pattern of activated MAPK within the PVN. These data are the first to suggest a differential coupling of MC4-R and may describe a mechanism through which the long-term and persistent behavioral actions of melanocortins are mediated.

Nitrotyrosine is widely recognized as a surrogate marker of up-regulated inducible NO synthase expression at sites of inflammation. However, the potential immunogenicity of autologous proteins containing nitrotyrosine has not previously been investigated. Herein, we used the I-E(K)-restricted T cell epitope of pigeon/moth cytochrome c (PCC/MCC(88-103)) to assess the ability of T cells to recognize ligands containing nitrotyrosine. Substitution of the single tyrosine (Y97) in PCC/MCC(88-103) with nitrotyrosine abrogates recognition by the MCC(88-103)-specific T cell hybridoma 2B4. CBA (H2(K)) mice immunized with MCC(88-103) or nitrated MCC(88-103) peptides produce T cell responses that are mutually exclusive. Transgenic mice that constitutively express PCC under the control of an MHC class I promoter are tolerant toward immunization with MCC(88-103), but exhibited a robust immune response against nitrated MCC(88-103). Analysis of T cell hybridomas specific for nitrated-MCC(88-103) indicated that subtle differences in TCR VDJ gene usage are sufficient to allow nitrotyrosine-specific T cells to escape the processes of central tolerance.

While the mechanism whereby the angiotensin II type 1 receptor (AT(1) receptor) activates its classical effector phospholipase C-beta (PLC-beta) has largely been elucidated, there is little consensus on how this receptor activates a more recently identified effector, the p42/44 mitogen-activated protein kinases (p42/44(MAPK)). Using transfected COS-1 cells, we investigated the activation of this signaling pathway at the receptor level itself. Previous mutational studies that relied on phosphoinositide turnover as an index of receptor activation have indicated that key residues in the second and seventh transmembrane domains participate in AT(1) receptor activation mechanisms. Thus, we introduced a variety of mutations-AT(1)[D74N], AT(1)[Y292F], AT(1)[N295S], and AT(1)[AT(2) TM7], which is composed of a chimeric substitution of the AT(1) seventh transmembrane domain with its AT(2) counterpart. These mutations that strongly diminished the receptor's ability to activate PLC-beta had little to no effect on its ability to activate p42/44(MAPK), which not only suggests that p42/44(MAPK) does not exclusively lie downstream of the G-protein G(q)/PLC-beta pathway but also indicates that more than one activation state may exist for the AT(1) receptor. The failure of a protein kinase C inhibitor to block AT(1) receptor activation of p42/44(MAPK) further corroborated evidence that the receptor's activation of p42/44(MAPK) is largely independent of the G(q)/PLC-beta/PKC pathway. Taken together, the experimental evidence strongly suggests that the mechanism whereby the AT(1) receptor activates p42/44(MAPK) is fundamentally different from that for PLC-beta, even at the level of the receptor itself.

Responsiveness of CD4+ T cells to the IFN-gamma-inducing cytokines IL-12 and IL-18 is generally thought to be acquired only after stimulation via the TCR. We report herein that stimulation of naive CD4+ T cells with high-dose IL-2 (1000 U/ml) renders these cells responsive to IL-12 and/or IL-18 without a requirement for TCR ligation. Naive CD4+CD62L+ Tcells from normal C57BL/6 mice or from DO11.10/Rag2(-/- )OVA-specific TCR-transgenic mice secreted substantial amounts of IFN-gamma when stimulated concurrently with high-dose IL-2 plus IL-12 or IL-18. mRNA encoding both chains of the IL-12 and the IL-18 receptors was expressed by CD4+ T cells after stimulation with high-dose IL-2. Furthermore, anti-CD3-induced IL-12/IL-18 responsiveness was fully abrogated in the presence of cyclosporin A whereas IL-2-induced IL-12/IL-18 responsiveness was not, reminiscent of the previously reported IL-12+IL-18 innate pathway of T cell activation. Lastly, after stimulation with IL-2+IL-12, naive CD4+ T cells from DO11.10/Rag2(-/- )mice exhibited polarization towards a Th1 phenotype (high IFN-gamma but no IL-4) during secondary stimulation with immobilized anti-CD3. We have coined the term "bystander polarization" to describe this phenomenon and we speculate that bystander polarization of naive CD4+ T cells may occur in vivo during strong antigen-specific immune responses.

A good in vitro model within which to investigate molecular interactions between feeding relevant neuropeptide systems has been lacking. Consequently, we began using reverse transcriptase-polymerase chain reaction (RT-PCR) to screen various neuronal cell lines for the presence of feeding relevant neuropeptides and receptors. N1E-115 murine neuroblastoma cells have emerged as an attractive candidate for further analysis because they contain mRNA for a variety of key systems implicated in the regulation of energy homeostasis., (Copyright 2002 Elsevier Science B.V.)

Chimeric AT1/AT2 angiotensin II (AngII) receptors in which the sixth and/or seventh transmembrane-spanning domains of the AT2 receptor were substituted into the AT1 receptor were used to investigate the activation mechanisms of the two receptor subtypes. Numerous reports have identified amino acid residues in the sixth and seventh transmembrane-spanning domains of the AT1 receptor involved in the intrareceptor activation mechanism following agonist binding. Many of these residues are not conserved in the AT2 receptor; the corresponding AT2 receptor residues are, in fact, disruptive of AngII-dependent activation when substituted into the AT1 receptor. Surprisingly, the chimeric AT1/AT2 receptors--which also lack these crucial AT1 residues--exhibited AngII-induced activation of phosphoinositide hydrolysis with efficacies and potencies similar to the wild-type AT1 receptor. Consistent with earlier reports, a AT1[Y292F] point mutant demonstrated greatly decreased agonist-induced activation of phosphoinositide hydrolysis. However, a AT1[Y292F/N295S] double-point mutant allowed for normal agonist-induced activation with a pharmacodynamic profile indistinguishable from the wild-type receptor. Despite amino acid dissimilarities, the same corresponding domains and even the same residue loci in both of the AngII receptor subtypes are equally able to mediate agonist-induced receptor activation. This suggests that these corresponding domains in the AT1 and the AT2 receptors are crucial to the activation mechanism, demonstrating greater structural flexibility than previously believed regarding AT1 receptor activation and supporting the possibility of a common activation mechanism for the two receptor subtypes.

Chimeric angiotensin II (AngII) receptors constructed of portions of the AT2 receptor substituted into the AT1 receptor revealed the AT2 third extracellular loop and seventh transmembrane-spanning domain as major determinants for the ability to bind and activate in response to the AT2 receptor-selective agonist CGP 42112A. Radioligand binding experiments showed that chimeric AngII receptors possessing the AT2 third extracellular loop and seventh transmembrane-spanning domain bound CGP 42112A with high affinity approaching that of the wild-type AT2 receptor. The presence of the AT2 third extracellular loop appeared sufficient for high-affinity CGP 42112A binding, which was further enhanced by the additional presence of the AT2 seventh transmembrane-spanning domain. Experiments with PD 123319, losartan, and [Sar1,Ile8]-AngII showed that increases in binding affinity associated with these domains were specific for CGP 42112A. Use of phosphoinositide hydrolysis as a functional index to measure activation of these chimeric AngII receptors further demonstrated that the AT2 seventh transmembrane-spanning domain was especially critical for CGP 42112A to act as an agonist. The absence of the AT2 seventh transmembrane-spanning domain prohibited CGP 42112A-induced activation of these receptors, even in the presence of high concentrations of CGP 42112A sufficient to saturate the binding sites. This study is the first to identify binding determinants of the AT2 receptor that are selective for CGP 42112A, and indicates that these determinants are at least partially distinct from those for the AT2-selective antagonist PD 123319. These differences may be a factor in the pharmacodynamic difference between these two ligands.

The angiotensin II (AngII) receptor family is comprised of two subtypes, type 1 (AT(1)) and type 2 (AT(2)). Although sharing low homology (only 34%), mutagenesis has identified some key residues that are conserved between both subtypes, including four extracellular cysteines. Previous AT(1) mutagenesis demonstrated that the cysteines form two disulfide bonds, one linking the first and second extracellular loops and another connecting the amino terminus to the third extracellular loop. The importance of these AT(1) disulfides in ligand binding is supported by the effect of dithiothreitol (DTT). DTT breaks disulfide bonds, thereby strongly inhibiting ligand binding in AT(1) receptors. Despite retaining the same cysteines, AT(2) receptor ligand binding is paradoxically enhanced by DTT. Thus, we constructed a series of AT(2) cysteine mutations, either individually or paired, to establish the role of the cysteines and the source of DTT's effects. The AT(2) cysteine mutants surprisingly confirmed that the cysteines form disulfide bonds in the same manner as in the AT(1) subtype. However, breaking the AT(2) disulfide bridges yielded two responses. As in AT(1) receptors, mutations disrupting the disulfide bond between the first and second extracellular loops reduced AT(2) binding by 4-fold. In contrast, mutations breaking the disulfide bridge between the amino terminus and the third extracellular loop increased AT(2) binding, mimicking DTT's effect on this subtype. Further analysis of AT(1)/AT(2) chimeric exchange mutants of these domains suggested that the AT(2) amino terminus and third extracellular loop may possess latent binding epitopes that are only uncovered after DTT exposure.

Recent studies demonstrate that cellular, molecular and morphological changes induced by stress in rats are accelerated when there is a pre-existing strain upon their already compromised adaptive responses to internal or external stimuli, such as may occur with uncontrolled diabetes mellitus. The deleterious actions of diabetes and stress may increase oxidative stress in the brain, leading to increases in neuronal vulnerability. In an attempt to determine if stress, diabetes or stress+diabetes increases oxidative stress in the hippocampus, radioimmunocytochemistry was performed using polyclonal antisera that recognize proteins conjugated by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE). Radioimmunocytochemistry revealed that HNE protein conjugation is increased in all subregions of the hippocampus of streptozotocin (STZ) diabetic rats, rats subjected to restraint stress and STZ diabetic rats subjected to stress. Such increases were not significant in the cortex. Because increases in oxidative stress may contribute to stress- and diabetes-mediated decreases in hippocampal neuronal glucose utilization, we examined the stress/diabetes mediated HNE protein conjugation of the neuron specific glucose transporter, GLUT3. GLUT3 immunoprecipitated from hippocampal membranes of diabetic rats subjected to stress exhibited significant increases in HNE immunolabeling compared to control rats, suggesting that HNE protein conjugation of GLUT3 contributes to decreases in neuronal glucose utilization observed during diabetes and exposure to stress. Collectively, these results demonstrate that the hippocampus is vulnerable to increases in oxidative stress produced by diabetes and stress. In addition, increases in HNE protein conjugation of GLUT3 provide a potential mechanism for stress- and diabetes-mediated decreases in hippocampal neuronal glucose utilization.

Angiotensin II (AngII) mediates its physiological actions through two receptor subtypes: the Type 1 (AT1) and Type 2 (AT2) receptors. The subtypes have identical affinities for AngII, while sharing only 34% homology. Mutagenesis has focused mainly on the AT1 receptor, identifying residues important for AngII binding. In contrast, relatively little is known of the binding mechanism of the AT2 receptor. It has been hypothesized that residues that are conserved between the two subtypes that have been shown to be important in the AT1 receptor may also contribute to AngII binding in the AT2 receptor as well. To test this hypothesis, the role of two conserved residues in the sixth transmembrane domain of the AT2 receptor in ligand binding were investigated: tryptophan 269 and aspartate 279. In contrast to the AT1 receptor, mutation of Trp269 in the AT2 receptor to an alanine had no effect on AngII binding, while mutation of Asp279 to alanine similarly impaired AngII binding in both receptors. However, the more sterically conservative substitution of Asp279 to asparagine in the AT2 receptor showed near wild type affinity. Based on this finding, we mutated Asp263 in the AT1 receptor to asparagine. Subsequent studies indicated that this more conservative mutation had no effect on AngII binding to the AT1 receptor. Collectively, these results demonstrate that although there may be commonalities in ligand binding between the AT1 and AT2 AngII receptors, there are also clear differences.

Key amino terminal residues in type 1 (AT1) angiotensin II (AngII) receptors are not conserved within type 2 (AT2) receptors. We therefore characterized amino terminal mutants that are transiently expressed in COS-3 membranes. AT2 amino terminal deletion drastically reduced affinity for AngII, suggesting its importance for this subtype. AT1-AT2 amino terminal exchanges retained wild type AngII affinities (Kd ranging from 3-5 nM), indicating compensation despite substantial sequence dissimilarities. Finally, binding of AT2 selective ligands (CGP42112A and PD123319) was not dependent on the amino terminus.

A fifth transmembrane domain lysine residue is conserved in both the type 1 (AT1) and type 2 (AT2) angiotensin II (AngII) receptors. This lysine (Lys199) is believed to play a critical role in peptide binding for the AT1 receptor. To evaluate its possible role in the AT2 receptor, the analogous AT2 residue (Lys199) was changed to glutamine. This mutation greatly reduced the affinity for both 125I-AngII and 125I-Sar1,Ile8-AngII and abolished binding to the non-peptide 125I-PD122979. These data indicate that despite a relatively low homology of 34%, some commonalities in the binding mechanism for AngII may exist between the two subtypes.

While much work has been done examining the ligand-binding characteristics of the AT1 receptor, very little attention has been focused on the AT2 receptor. Both receptors bind angiotensin II (AngII) with identical affinities, but share only 34% homology. Although it is tempting to assume that conserved residues between the two subtypes are responsible for the binding of AngII, there is little data to support this view. To determine the commonalities in ligand binding of the AT1 and AT2 receptors, we have chosen several conserved extracellular amino acids which have been shown to be important in AngII binding [1,2] to the AT1 receptor for mutational studies of the AT2 receptor. Specifically, we have mutated tyrosine108 in extracellular loop 1 (ECL1), arginine182 in ECL2, and aspartate297 in ECL3 of the AT2 receptor in order to determine their contribution to AngII binding. In the AT2 receptor, mutation of tyrosine108 to an alanine resulted in a receptor with wild-type binding for AngII, while mutation of either arginine182 or aspartate297 drastically impaired AngII binding ( > 100 nM). These results demonstrate both similarities as well as clear differences between receptor subtypes in the contributions to AngII binding of several conserved extracellular amino acid residues.

Homology-based PCR was used to isolate angiotensin II type 2 (AT2) receptor cDNA from murine neuroblastoma N1E-115 cells. Despite subtle differences in the nucleotide sequence (the N1E-115 clone coded for Phe133 as TTC and Gln326 as CAG; base substitutions are in bold-italics), the AT2 receptor protein was identical to other reported murine AT2 clones. When transfected into COS-1 cells, the expressed AT2 receptor displayed high affinity for AngII and for AT2-selective compounds, GTP gamma S-insensitive agonist binding and enhanced agonist binding by dithiothreitol. Previously, we have demonstrated that N1E-115 cells possess two distinct subpopulations of AT2 receptors, defined as peak I and peak III receptors, that can be separated by heparin-sepharose chromatography. The two subpopulations differ pharmacologically, biochemically and immunologically. The binding properties of the cloned AT2 receptor closely resembled that of peak III receptors. Moreover, antisera raised against peak I AT2 receptors failed to immunoreact to either peak III receptors or cloned AT2 receptors expressed in COS-1 cells. Collectively, these data suggest that the cloned AT2 receptor is identical to peak III receptors from N1E-115 cells and that a novel AT2 receptor (peak I) remains to be cloned.

The contributions of managed care pharmacists who participate in clinical rounds at contract hospitals are described. A program in which pharmacists employed by Kaiser Permanente go on rounds at two contract hospitals as part of a multidisciplinary Kaiser team was implemented in January 1995. The rounding pharmacist reviews patients' charts, assesses optimal drug therapy, intervenes proactively, identifies candidates for home i.v. therapy, facilitates patient discharge, and provides on-the-spot drug information. Other services include therapeutic drug monitoring, dosage-adjustment recommendations, counseling, consultations, detecting adverse drug reactions, and education. The pharmacist documents all services and interventions, and their impact. At one hospital, a 500-bed institution, there were 1097 recommendations for intervention between April 1995 and May 1996. More than 99% of these recommendations were accepted. Over the 14-month period, the Kaiser team saved an estimated $523,907 and cost an estimated $57,643 (not counting benefits) in pharmacist participation. Managed care pharmacists on rounds at contract hospitals influenced enrolled patients' pharmacotherapy and reduced the plan's costs.

Evidence continues to accumulate that strengthens the proposal of heterogeneity within both the AT1 and the AT2 receptor subtypes. Pharmacologic, biochemical and immunological studies of AT2 receptors expressed in N1E-115 cells strengthen the hypothesis of AT2 receptor heterogeneity. However, it is important to reassess these studies, especially in terms of how these results correlate with other reports of AT2 receptor heterogeneity. For example, AT2 receptor immunoreactivity was absent in some neuronal regions which have previously been proposed to express the AT2 receptor subtype. In particular, AT2 receptor staining was not seen in the inferior olive, a region which is known to express a high density of AT2 receptors. Upon first examination, these results were somewhat troubling. However, when compared with earlier reports, these results should not have been unexpected. For instance, Tsutsumi and Saaverdra previously have shown that AT2 receptors in the locus coeruleus are sensitive to the actions of guanine nucleotides, while AT2 receptors in the inferior olive are insensitive (21). These antisera were raised against a population of AT2 receptors which are sensitive to GTP gamma S and therefore, the lack of AT2 receptor staining in the inferior olive, as well as the presence of AT2 receptor immunoreactivity in the locus coeruleus, confirms and extends these earlier reports. In addition the AT2 receptors expressed in the locus coeruleus have been shown to be functionally distinct from AT2 receptors in the inferior olive. In this regard, Ang II has been shown to depress glutamate-induced EPSPs in the locus coeruleus, an effect which is mediated through the AT2 receptor (19). Conversely, AT2 receptors have been shown to increase the firing rate of neurons in the inferior olive (20). Collectively, these results would predict that staining should be absent in the inferior olive using these AT2-directed antisera. Indeed, in view of these earlier physiological and pharmacological studies, the presence of AT2 receptor immunoreactivity in the inferior olive would have been surprising. The most convincing example of AT2 receptor heterogeneity is the characterization of AT2 receptors present in N1E-115 cells. Separation of solubilized N1E-115 membranes by heparin-Sepharose chromatography generates two populations of AT2 receptors which are pharmacologically and biochemically distinct. In particular, CGP42112A was approximately 2 orders of magnitude more selective for Peak III AT2 receptors than was PD123319. Binding activity of Peak I and Peak III AT2 receptor populations also differed in their responses to GTP gamma S and DTT treatment. Lastly, the AT2-directed antisera, raised against the Peak I population of AT2 receptors, were not able to immunodetect the Peak III population of AT2 receptors in immunoblot analysis, or immunoprecipiatate AT2 binding activity from Peak III material. Pharmacological, biochemical and immunological analysis of the AT2 receptor clone isolated from N1E-115 cells revealed that it has the identical characteristics or properties of the Peak III receptor. The AT2 receptor isolated from N1E-115 cells exhibited a similar pharmacology as the Peak III AT2 receptor, in that CGP42112A was more effective at displacing 125I-Ang II binding activity than was PD123319. The AT2 receptor clone was also shown to be insensitive to the actions of GTP gamma S, as well as demonstrated increased binding activity in the presence of DTT, identical to the Peak III AT2 receptor. Lastly, immunoblot analysis of membranes prepared from COS-1 cells transfected with the AT2 receptor cDNA from N1E-115 cells did not demonstrate any immune-specific bands with the AT2-directed antisera. Characterization of an AT2 receptor cDNA isolated from N1E-115 cells reveals that this clone is identical to the Peak III type of AT2 receptor.

The diverse actions of angiotensin II (AngII) are mediated by cell surface receptors. Molecular cloning techniques have identified two distinct subtypes of AngII receptors referred to as AT1 and AT2. It is now well accepted that multiple forms of the AT1 receptor exist, but similar diversity of the AT2 subtype has not been conclusively demonstrated. Nonetheless, several converging lines of evidence do suggest that multiple AT2 receptors may be present in brain and cultured neuron-like cells lines. For instance, some AT2 receptors are regulated by guanine nucleotides and sulfhydryl-reducing agents, whereas others are insensitive. AT2 receptor populations also exhibit differing pharmacological profiles particularly with respect to their affinity for peptidic and non-peptidic ligands. Moreover, a recently developed anti-AT2 polyclonal antisera reveals a unique pattern of immunohistochemical staining in brain and it does not immunoreact with the recently cloned AT2 receptor. Collectively, these results support the hypothesis of multiple AT2 receptors at least within the CNS. Future studies should reveal whether these putative AT2 receptor subtypes result from unique genes or cell-specific post-translational modifications of a single gene product.

Previous work has demonstrated that crosslinking of [125I]AngII to CHAPS solubilized angiotensin Type 2 receptors (AT2) in N1E-115 neuroblastoma cells identifies two radiolabeled proteins of 110 and 66 kDa. Similarly, affinity purification of AT2 receptors using AngII yields two proteins of 110 and 66 kDa. In the present study, anti-AT2 receptor antisera were used to examine the relationship between these two proteins. Agonist treatment (AngII) of intact cells increased the 110 kDa band while decreasing the 66 kDa protein. In intact or solubilized membranes, the ratio of 110 kDa/66 kDa proteins was significantly higher in the presence of an agonist and substantially lower with the antagonist Sar1,Ile8-AngII, suggesting that AngII stabilizes a large 110 kDa multimeric complex that may include the 66 kDa protein. To directly examine this hypothesis, anti-AT2 antisera were further purified against either the 110 or 66 kDa proteins. Both purified antibodies displayed crossreactivity with the two proteins. Moreover, when harshly reduced and denatured, the 110 kDa protein released a prominent immunoreactive 66 kDa protein, as well as other smaller proteins. Collectively, these results suggest that the 110 kDa protein consists, in part, of the 66 kDa protein and, as such, that an AT2 receptor subtype may exist as a multimeric complex that is stabilized by agonist occupancy.

Recently developed antisera selective for angiotensin Type 2 (AT2) receptors were used to localize AT2 receptors in rat brain by immunohistochemistry. While the results from these experiments were largely consistent with previous autoradiographic and radioligand binding analyses of AT2 receptor populations in brain, there were also some notable differences in the distribution of immunoreactivity. More specifically, in agreement with previous studies, AT2 antisera detected apparent receptor populations in the locus coeruleus and the bed nucleus of the accessory olfactory tract, whereas AT2 receptor-immunoreactivity in the cerebellum was primarily associated with the Purkinje cell layer and the deep cerebellar nuclei rather than the molecular layer as has been previously reported in autoradiographic studies. Other regions with prominent immune-staining included all subfields of the hippocampus, which had been previously reported to contain exclusively AT1 receptors. Limbic structures such as the amygdala, thalamic areas such as the rhomboid thalamic nucleus, the paraventricular thalamic nucleus, hypothalamic areas such as the paraventricular hypothalamic nucleus, and the supraoptic nucleus also exhibited prominent AT2-immunoreactivity. In the paraventricular hypothalamic nucleus, AT2 receptor staining appeared to be associated primarily with the magnocellular neurons. In all regions examined, AT2 receptor immunoreactivity was associated with the cytoplasm and cell membrane and was not localized within the nucleus. Collectively, these results confirm and extend the neuroanatomical resolution of previous autoradiographic studies as well as identify new AT2 receptor populations in rat brain.

This longitudinal survey study of 557 college women used the new Eating Pathology Scale to classify respondents as nondieters, casual dieters, intensive dieters, dieters at risk, and bulimic. Shifts in the severity of dieting behavior over a 6-month period occurred primarily between adjacent scale categories. While new cases of bulimia were drawn from intensive dieters and dieters at risk, those women who no longer met DSM-III-R criteria for bulimia nervosa continued to engage in bulimic behavior.

The murine neuroblastoma N1E-115 cell line possesses a high density of angiotensin II (AngII) receptors that can be solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. These solubilized binding sites exhibited high affinity for CGP-42112A and not Losartan, indicating that they were of the AT2 subtype. However, displacement of 125I-AngII with the AT2 nonpeptide antagonist PD-123319 resulted in a biphasic curve, suggesting heterogeneity of the AT2 receptor population in N1E-115 cells. In support of this view, separation of two receptor populations was accomplished with heparin-Sepharose chromatography. More specifically, three distinct protein peaks eluted from the heparin-Sepharose column, two of which bound 125I-AngII with high affinity and saturability. One of these binding peaks (peak I) eluted rapidly and represented approximately 80% of the total binding activity, whereas the remaining binding activity was contained within a second peak (peak III) that required the addition of 1.5 M NaCl for its complete elution. Pharmacological analysis revealed that both peaks of binding activity were exclusively AT2 receptors insofar as they exhibited high affinity for CGP-42112A and little or no affinity for the AT1-selective antagonist Losartan. However, whereas the nonpeptidic AT2-selective antagonist PD-123319 completely displaced the binding of 125I-AngII from peak I in a monophasic fashion (IC50 = 9.1 +/- 4.1 nM; mean +/- SEM; n = 3), PD-123319 was much less effective in displacing 125I-AngII from peak III (IC50 = 196 +/- 27 nM; mean +/- SEM; n = 3). Treatment of individual peaks with the reducing agent dithiothreitol caused a large increase in 125I-AngII specific binding in peak III, whereas a decrease in binding was observed in peak I. Moreover, GTP gamma S significantly reduced high-affinity agonist binding in peak I but not peak III, further suggesting heterogeneity in the AT2 receptor family. Finally, immunoblotting studies with polyclonal antisera raised against peak I specifically detected two proteins of 110 and 66 kDa, as is true in crude solubilized membranes, whereas no immunospecific proteins were detected in peak III. These same antisera immunoprecipitated 125I-AngII binding activity in peak I but were ineffective in peak III. Collectively, these results suggest that heparin-Sepharose chromatography can efficiently separate two pharmacologically, biochemically and immunologically distinct populations of AT2 receptors.

The murine neuroblastoma N1E-115 cell line possesses type 1 and type 2 angiotensin II (AngII) receptor subtypes. In vitro differentiation of these cells substantially increases the density of the AT2-receptor subtype, whereas the density of the AT1 receptors remains unchanged. In the present study, we report that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) selectively solubilized AT2 receptors from N1E-115 cell membranes and that these receptors could be purified further to near homogeneity by affinity chromatography. More specifically, the presence of an agonist (AngII) during affinity purification of AT2 receptors resulted in the elution of high (110-kDa) and low (66-kDa) molecular mass proteins as determined by gel electrophoresis under nonreducing conditions. In contrast, when the nonselective antagonist Sar1,Ile8-AngII was used during purification, only the lower 66-kDa protein was observed. Affinity purification in the presence of the peptide and nonpeptide AT2-receptor antagonists CGP42112A and PD123319 also resulted in elution of the same 66-kDa protein, but unlike that in the presence of Sar1,Ile8-AngII, some of the high molecular weight site was observed as well. On the other hand, Losartan, an AT1-receptor antagonist, was completely ineffective in eluting any AngII receptors from the affinity column, further confirming their AT2 identity. After agonist elution, the 110-kDa band dissociated into two low molecular mass bands of 66 kDa and 54 kDa when sodium dodecyl sulfate-gel electrophoresis was run under reducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Murine neuroblastoma N1E-115 cells are a useful system in which to study neuronal angiotensin II (AngII) receptors. N1E-115 cells possess both type 1 (AT1) and type 2 (AT2) AngII receptor subtypes, as does mammalian brain. AT2 receptors in brain or N1E-115 cells can be solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. In the present study, heparin-Sepharose chromatography was used to partially purify solubilized N1E-115 membranes to produce an enriched population of AT2 receptors. Subsequently, an eluted peak, containing the majority of AT2 binding activity, was used as an immunogen in the development of protein-directed polyclonal antibodies. The antibodies specifically detected immunoreactive proteins of approximately 110 and 66 kDa in both solubilized N1E-115 cells, as well as the original protein material that eluted from the heparin-Sepharose column, whereas no such immunoreactivity was detected in a kidney epithelial cell line that lacks any specific 125I-labeled AngII (125I-AngII) binding activity. Moreover, the antibodies immunoreacted with affinity-purified AT2 receptors. These antibodies were also able to immunoprecipitate AT2 receptors from solubilized N1E-115 cells, as revealed by the pharmacologic profile of 125I-AngII binding to the precipitated protein. Similarly, the antibodies were able to immunoprecipitate a 66-kDa protein that had been covalently crosslinked with 125I-AngII by use of the homobifunctional crosslinker dithiobis(succinimidyl propionate). Collectively, these results demonstrate the development of a specific AT2 receptor antibody that may be used to further characterize this receptor subtype at both the cellular and molecular levels.