Normal-mode oscillations for the circular and dipolar states of a filled hexagonal magnetic dipole cluster.

We analyze the rotational dynamics of six magnetic dipoles of identical strength at the vertices of a regular hexagon with a variable-strength dipole in the center. The seven dipoles spin freely about fixed axes that are perpendicular to the plane of the hexagon, with their dipole moments directed parallel to the plane. Equilibrium dipole orientations are calculated as a function of the relative strength of the central dipole. Small-amplitude perturbations about these equilibrium states are calculated in the absence of friction and are compared with analytical results in the limit of zero and infinite central dipole strength. Normal modes and frequencies are presented. Bifurcations are seen at two critical values of the central dipole strength, with bistability between these values.

Pharyngeal Mucosal Margin Vessel Counts Predict Pharyngocutaneous Fistula in Salvage Laryngectomy.

OBJECTIVE: We evaluated vessel counts in the pharyngeal mucosal margins of patients who underwent salvage laryngectomy to establish whether mucosal vascularity might predict fistula risk. STUDY DESIGN: Retrospective cohort. SETTING: Tertiary Medical Center. METHODS: Patients who underwent salvage total laryngectomy at our institution between 1999 and 2015 were identified. Pharyngeal mucosal margins from laryngectomy specimens were evaluated histologically for each patient, and vessel counts were performed on 5 ×10 images. The primary outcome measure was fistula within 30 days of surgery and mean vessel counts were assessed as the principle explanatory variable. RESULTS: Seventy patients were included and 40% developed a postoperative fistula. There was a large difference in the mean vessel count in patients who did develop fistula (48.6 vessels/×10 field) compared to those who did not (34.7 vessels/×10 field). A receiver operative characteristic curve found that a cutoff value of 33.9 vessels/×10 field provided a sensitivity of 75% and specificity of 62% to predict the likelihood of fistula occurrence (area under the curve = 0.71, 95% confidence interval [CI]: 0.59-0.83). In a binary logistic regression, patients with vessel counts greater than 33.9 had a 5-fold increased risk of developing fistula (95% CI: 1.8-16.45). Histologically, vessels in the pharyngeal mucosa of patients who developed fistulas were more disorganized. CONCLUSION: After salvage laryngectomy, patients with higher mean mucosal margin vessel counts are at increased risk of fistula. The mechanism is unknown, but the disorganization of the vasculature may contribute to poor wound healing. Vessel counting may allow for fistula risk stratification and guide postoperative care.

Readiness of Medical Students to Care for Diverse Patients: A Validated Assessment of Cross-Cultural Preparedness, Skills, and Curriculum.

Introduction: Effective cross-cultural care is foundational for mitigating health inequities and providing high-quality care to diverse populations. However, medical school teaching practices vary widely, and learners have limited opportunities to develop these critical skills. To understand the current state of cross-cultural education and to identify potential opportunities for improvement, we disseminated a validated survey instrument among medical students at a single institution. Methods: Learners across 4 years of medical school participated in the cross-cultural care assessment, using a tool previously validated with resident physicians and modified for medical students. The survey assessed medical student perspectives on (1) preparedness, (2) skillfulness, and (3) educational curriculum and learning environment. Cross-sectional data were analyzed by class year, comparing trends between school years. Results: Of 700 possible survey responses, we received 260 (37% response rate). Fourth-year students had significantly higher scores than first-year students (p<0.05) for 7 of 12 preparedness items and 4 of 9 skillfulness items. Less than 50% of students indicated readiness to deliver cross-cultural care by their fourth year in 9 of 12 preparedness items and 6 of 9 skillfulness items. Respondents identified inadequate cross-cultural education as the primary barrier. Discussion: Medical students reported a lack of readiness to provide cross-cultural care, with self-assessed deficiencies persisting through the fourth year of medical school. Medical educators can use data from the cross-cultural care survey to longitudinally assess students and enhance curricular exposures where deficiencies exist. Optimizing cross-cultural education has the potential to improve the learning environment and overall patient care.

Role of the otologist/neurotologist in managing auricular and periauricular cutaneous malignancies: A 10-year otologic oncology experience.

Objective: Auricular/periauricular cutaneous malignancies can be challenging to manage surgically due to the complex anatomy of the region. Otologists/neurotologists have unique skillsets that are well-suited to surgically treat these patients. We aim to highlight the role of otologists and neurotologists in providing surgical care of patients with auricular and periauricular malignancies by describing the experience of a single fellowship-trained neurotologist over a 10-year period. Methods: Retrospective chart review of 387 patients with auricular and periauricular malignancy treated by a single neurotologist between 2012 and 2022 was completed. Tumor histology and procedures performed for each patient were extracted. Additional data was collected for a subset of 84 patients with complex cases requiring selective neck dissection, parotidectomy, lateral temporal bone resection, regional advancement or rotational flap reconstruction, and/or free tissue transfer reconstruction. Results: Within the series of 387 patients, squamous cell carcinoma was the most common histology (42.6%, n = 165), followed by basal cell carcinoma (40.8%, n = 158), and melanoma (9.8%, n = 38). Common surgical procedures included wide local excision (61.8%, n = 239), partial/sub-total auriculectomy 18.3% (n = 71), or total auriculectomy 5.2% (n = 20). Within the 84-patient subset, median age at diagnosis was 71.9 years. Dermatologists provided most patient referrals (50.0%, n = 42). Most common tumor locations included: auricular (58.3%, n = 49), pre-auricular (21.4%, n = 18), and parotid (27.4%, n = 23). Revision surgery occurred in 22.6% of cases (n = 19), of which 26.3% (n = 5) for positive margins and 31.6% (n = 6) for recurrence. Mean follow-up was 22.8 months. Disease-specific 5-year survival was 91%. Conclusions: We demonstrate the feasibility of an otologist/neurotologist incorporating the surgical management of auricular and periauricular malignancies into their practice. Level of Evidence: 4.

Verrucous Carcinoma of the Foot: A Retrospective Study of 19 Cases and Analysis of Prognostic Factors Influencing Recurrence.

This study sought to evaluate a cohort of patients with verrucous carcinoma of the foot with special focus on 5 cases of locally recurrent tumors despite negative margins. Nineteen cases of verrucous carcinoma of the foot were identified through the University of Michigan (Ann Arbor, Michigan) pathology database from 1995 to 2019 and were included in demographic and clinical presentation analyses. Sixteen cases were treated at the University of Michigan and are included in the treatment analyses. A review of medical records was conducted to characterize clinical, surgical, and pathologic features. Recurrent cases were found to have a predilection for nonglabrous skin of the foot and great toe. Otherwise, there was little to differentiate outcomes between recurrent and nonrecurrent groups based on demographic, clinical, surgical, or histopathologic data. Recurrent tumors regrew locally and were not associated with histologic progression to conventional squamous cell carcinoma. Verrucous carcinoma of the nonglabrous surface of the foot should have a higher suspicion for possible local recurrence. Recurrence occurs within months of treatment, deserves early biopsy, and warrants aggressive re-treatment. Future directions should include greater examination of pathologic features and genetic markers to improve management of verrucous carcinoma of the foot.

Sudden Sensorineural Hearing Loss: A Diagnostic and Therapeutic Emergency.

The family physician's role in recognizing and managing sudden sensorineural hearing loss (SSNHL) is crucial. A recently updated otolaryngologic clinical practice guideline has been released for this emergency syndrome, but dissemination is limited to a specialty journal. As a result, the guidelines may not be widely available in the primary care setting where patients often present. We provide this focused review to clarify and disseminate SSNHL guidelines for the frontline family physician.

The Clarion Call of the COVID-19 Pandemic: How Medical Education Can Mitigate Racial and Ethnic Disparities.

Public health crises palpably demonstrate how social determinants of health have led to disparate health outcomes. The staggering mortality rates among African Americans, Native Americans, and Latinx Americans during the COVID-19 pandemic have revealed how recalcitrant structural inequities can exacerbate disparities and render not just individuals but whole communities acutely vulnerable. While medical curricula that educate students about disparities are vital in rousing awareness, it is experience that is most likely to instill passion for change. The authors first consider the roots of health care disparities in relation to the current pandemic. Then, they examine the importance of salient learning experiences that may inspire a commitment to championing social justice. Experiences in diverse communities can imbue medical students with a desire for lifelong learning and advocacy. The authors introduce a 3-pillar framework that consists of trust building, structural competency, and cultural humility. They discuss how these pillars should underpin educational efforts to improve social determinants of health. Effecting systemic change requires passion and resolve; therefore, perseverance in such efforts is predicated on learners caring about the structural inequities in housing, education, economic stability, and neighborhoods-all of which influence the health of individuals and communities.

Effects of Moringa Extract on Aminoglycoside-Induced Hair Cell Death and Organ of Corti Damage.

HYPOTHESIS: Moringa extract, a naturally occurring anti-oxidant, protects against aminoglycoside-induced hair cell death and hearing loss within the organ of Corti. BACKGROUND: Reactive oxygen species (ROS) arise primarily in the mitochondria and have been implicated in aminoglycoside-induced ototoxicity. Mitochondrial dysfunction results in loss of membrane potential, release of caspases, and cell apoptosis. Moringa extract has not previously been examined as a protective agent for aminoglycoside-induced ototoxicity. METHODS: Putative otoprotective effects of moringa extract were investigated in an organotypic model using murine organ of Corti explants subjected to gentamicin-induced ototoxicity. Assays evaluated hair cell loss, cytochrome oxidase expression, mitochondrial membrane potential integrity, and caspase activity. RESULTS: In vitro application of moringa conferred significant protection from gentamicin-induced hair cell loss at dosages from 25 to 300 µg/mL, with dosages above 100 µg/mL conferring near complete protection. Assays demonstrated moringa extract suppression of ROS, preservation of cytochrome oxidase activity, and reduction in caspase production. CONCLUSION: Moringa extract demonstrated potent antioxidant properties with significant protection against gentamicin ototoxicity in cochlear explants.

Dual binding capacity of mucosal immunoblasts to mucosal and synovial endothelium in humans: dissection of the molecular mechanisms.

Lymphocytes continuously migrate throughout the body in search of antigens. Virgin lymphocytes recirculate freely between the blood and different lymphatic organs, whereas immunoblasts extravasate preferentially into sites similar to those where they initially responded to antigen. Tissue-specific extravasation of lymphocytes is largely controlled by distinct lymphocyte surface receptors that mediate lymphocyte binding to high endothelial venules (HEV). In the present study, the molecular mechanisms determining the specificity of human mucosal (lamina propria) lymphocyte binding to different endothelial recognition systems were analyzed. Mucosal immunoblasts adhered five times better than small mucosal lymphocytes to mucosal HEV. Importantly, mucosal immunoblasts also bound to synovial HEV almost as efficiently as to mucosal HEV, but they did not adhere to peripheral lymph node HEV. To study the impact of different homing-associated molecules in this dual endothelial binding, we used a gut-derived T cell line and freshly isolated mucosal immunoblasts. Both cell types expressed integrins alpha 4, beta 1, beta 7, and lymphocyte function associated antigen 1 (LFA-1), and were CD44 positive, but practically L-selectin negative. Binding of mucosal immunoblasts to mucosal HEV was almost completely abolished by pretreatment with anti-beta 7 monoclonal antibodies, but it was independent of alpha 4/beta 1 function. In contrast, alpha 4/beta 1 partially mediated immunoblast adherence to synovial HEV, whereas alpha 4/beta 7 had only a minor role in adherence of blasts at this site. CD44 and LFA-1 contributed to HEV-binding both in mucosa and synovium. Taken together, this is the first report that demonstrates a critical role for alpha 4/beta 7 in the binding of gut lymphocytes to mucosal venules in humans. Moreover, a hitherto unknown interaction between mucosal effector cells and synovial endothelial cells was shown to be only partially mediated by the currently known homing receptors. The dual endothelial binding capacity of mucosal blasts may help to explain the pathogenesis of reactive arthritis not uncommonly associated with inflammatory and infectious bowel disease.

The alpha 4 integrin chain is a ligand for alpha 4 beta 7 and alpha 4 beta 1.

The heterodimeric alpha 4 integrins alpha 4 beta 7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-1) and alpha 4 beta 1 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-1, which binds to alpha 4 beta 1 and alpha 4 beta 7, and the mucosal addressin cell adhesion molecule (MAdCAM)-1, which binds to alpha 4 beta 7. Here we show that the alpha 4 chain of these integrins can itself serve as a ligand. The alpha 4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-alpha 4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively alpha 4 beta 7 (TK-1) or alpha 4 beta 1 (L1-2) both bound avidly, whereas alpha 4-negative cells did not. A soluble 34-kD alpha 4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to alpha 4 ligands. It has been shown that alpha 4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HepII/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of alpha 4. A synthetic LDV-containing alpha 4-derived oligopeptide supports alpha 4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD alpha 4 chain fragment. Our results suggest that alpha 4 beta 7 and alpha 4 beta 1 integrins may be able to bind to the alpha 4 subunit on adjacent cells, providing a novel mechanism for alpha 4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses.

Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: Epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity.

The immune system has evolved specialized cellular and molecular mechanisms for targeting and regulating immune responses at epithelial surfaces. Here we show that small intestinal intraepithelial lymphocytes and lamina propria lymphocytes migrate to thymus-expressed chemokine (TECK). This attraction is mediated by CC chemokine receptor (CCR)9, a chemoattractant receptor expressed at high levels by essentially all CD4(+) and CD8(+) T lymphocytes in the small intestine. Only a small subset of lymphocytes in the colon are CCR9(+), and lymphocytes from other tissues including tonsils, lung, inflamed liver, normal or inflamed skin, inflamed synovium and synovial fluid, breast milk, and seminal fluid are universally CCR9(-). TECK expression is also restricted to the small intestine: immunohistochemistry reveals that intense anti-TECK reactivity characterizes crypt epithelium in the jejunum and ileum, but not in other epithelia of the digestive tract (including stomach and colon), skin, lung, or salivary gland. These results imply a restricted role for lymphocyte CCR9 and its ligand TECK in the small intestine, and provide the first evidence for distinctive mechanisms of lymphocyte recruitment that may permit functional specialization of immune responses in different segments of the gastrointestinal tract. Selective expression of chemokines by differentiated epithelium may represent an important mechanism for targeting and specialization of immune responses.

Human G protein-coupled receptor GPR-9-6/CC chemokine receptor 9 is selectively expressed on intestinal homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required for thymus-expressed chemokine-mediated chemotaxis.

TECK (thymus-expressed chemokine), a recently described CC chemokine expressed in thymus and small intestine, was found to mediate chemotaxis of human G protein-coupled receptor GPR-9-6/L1.2 transfectants. This activity was blocked by anti-GPR-9-6 monoclonal antibody (mAb) 3C3. GPR-9-6 is expressed on a subset of memory alpha4beta7(high) intestinal trafficking CD4 and CD8 lymphocytes. In addition, all intestinal lamina propria and intraepithelial lymphocytes express GPR-9-6. In contrast, GPR-9-6 is not displayed on cutaneous lymphocyte antigen-positive (CLA(+)) memory CD4 and CD8 lymphocytes, which traffic to skin inflammatory sites, or on other systemic alpha4beta7(-)CLA(-) memory CD4/CD8 lymphocytes. The majority of thymocytes also express GPR-9-6, but natural killer cells, monocytes, eosinophils, basophils, and neutrophils are GPR-9-6 negative. Transcripts of GPR-9-6 and TECK are present in both small intestine and thymus. Importantly, the expression profile of GPR-9-6 correlates with migration to TECK of blood T lymphocytes and thymocytes. As migration of these cells is blocked by anti-GPR-9-6 mAb 3C3, we conclude that GPR-9-6 is the principal chemokine receptor for TECK. In agreement with the nomenclature rules for chemokine receptors, we propose the designation CCR-9 for GPR-9-6. The selective expression of TECK and GPR-9-6 in thymus and small intestine implies a dual role for GPR-9-6/CCR-9, both in T cell development and the mucosal immune response.

Head and Neck Reconstruction in the Vessel Depleted Neck.

Microvascular free tissue transfer has revolutionized reconstruction and subsequently functional outcomes in the head and neck, but requires suitable recipient vessels for successful results. Recipient vessels can be significantly compromised by prior surgery, radiation therapy, or existing and/or underlying vascular disease in the neck. When further microvascular reconstruction is required in the vessel-depleted neck, identification of appropriate vessels for anastomosis can be difficult and can present complex decisions for the surgeon as well as the patient. In this article, we review the available literature on the vessel depleted neck and the possible vessel options. We present critical strategies for preoperative treatment planning and vessel selection in these patients. We also discuss the benefits and limitations of arterial and venous options while commenting on our unique institution's experiences. The external carotid branches as well as the available subclavian artery branches are presented in detail. The venous anatomy is also described, with particular focus on the accompanying veins and cephalic vein. We provide guidance on the selection and modification of free flaps to achieve the greatest function and cosmetic outcomes in the vessel depleted neck. Our collection of advanced management techniques will provide surgeons with more options to manage the complexity of the vessel depleted neck, and to further help patients understand the risk and benefits of these selections.

Airway Management for Endoscopic Laryngotracheal Stenosis Surgery During COVID-19.

The novel coronavirus disease 2019 (COVID-19) pandemic presents unique challenges for surgical management of laryngotracheal stenosis. High viral concentrations in the upper aerodigestive tract, the ability of the virus to be transmitted by asymptomatic carriers and through aerosols, and the need for open airway access during laryngotracheal surgery create a high-risk situation for airway surgeons, anesthesiologists, and operating room personnel. While some surgical cases of laryngotracheal stenosis may be deferred, patients with significant airway obstruction or progressing symptoms often require urgent surgical intervention. We present best practices from our institutional experience for surgical management of laryngotracheal stenosis during this pandemic, including preoperative triage, intraoperative airway management, and personal protective measures.

Rules of chemokine receptor association with T cell polarization in vivo.

Current concepts of chemokine receptor (CKR) association with Th1 and Th2 cell polarization and effector function have largely ignored the diverse nature of effector and memory T cells in vivo. Here, we systematically investigated the association of 11 CKRs, singly or in combination, with CD4 T cell polarization. We show that Th1, Th2, Th0, and nonpolarized T cells in blood and tissue can express any of the CKRs studied but that each CKR defines a characteristic pool of polarized and nonpolarized CD4 T cells. Certain combinations of CKRs define populations that are markedly enriched in major subsets of Th1 versus Th2 cells. For example, although Th0, Th1, and Th2 cells are each found among blood CD4 T cells coordinately expressing CXCR3 and CCR4, Th1 but not Th2 cells can be CXCR3(+)CCR4(-), and Th2 but only rare Th1 cells are CCR4(+)CXCR3(-). Contrary to recent reports, although CCR7(-) cells contain a higher frequency of polarized CD4 T cells, most Th1 and Th2 effector cells are CCR7(+) and thus may be capable of lymphoid organ homing. Interestingly, Th1-associated CKRs show little or no preference for Th1 cells except when they are coexpressed with CXCR3. We conclude that the combinatorial expression of CKRs, which allow tissue- and subset-dependent targeting of effector cells during chemotactic navigation, defines physiologically significant subsets of polarized and nonpolarized T cells.

Analgesic efficacy of the cyclooxygenase-inhibiting nitric oxide donor AZD3582 in postoperative dental pain: Comparison with naproxen and rofecoxib in two randomized, double-blind, placebo-controlled studies.

OBJECTIVE: This study assessed the analgesic efficacy of single doses of 4-(nitrooxy)butyl-(2S)-2-(6-methoxy-2-naphthyl) propanoate (AZD3582) in acute postoperative dental pain after the removal of an impacted mandibular third molar (ie, wisdom tooth). METHODS: Two randomized, placebo-controlled, double-blind studies were performed. In a dose-finding study, 242 patients were randomized to AZD3582 375, 750, 1500, or 2250 mg (n = 41, 37, 42, and 41, respectively); naproxen 500 mg (n = 39); or placebo (n = 42). In a comparator study, 282 patients were randomized to AZD3582 500 mg (n = 78) or 750 mg (n = 83), rofecoxib 50 mg (n = 80), or placebo (n = 41). Primary outcomes included time to rescue medication, time to pain relief, and mean pain intensity difference (MPID), as well as safety profile. Pain was rated on a visual analog scale. RESULTS: In the dose-finding study, 52% (126/242) were women; the mean (SD) age was 25.1 (4) years, mean weight was 69.0 kg, and the mean (SD) body mass index (BMI) was 23.7 (3) kg/m2. In the comparator study, 58% (164/282) were women; the mean (SD) age was 27 (6.4) years, mean weight was 71 kg, and mean (SD) BMI was 24.2 (3) kg/m2. In the dose-finding study, the AZD3582 750-, 1500-, and 2250-mg groups were superior to placebo in the primary variables "time to rescue medication (0-8 hours)" (hazard ratios [HRs] [95% CIs], 0.17 [0.07-0.42], P < 0.003; 0.23 [0.11-0.50], P < 0.001; and 0.15 [0.06-0.36], P < 0.001, respectively), "time to meaningful pain relief" (HRs [95% CIs], 3.42 [1.87-6.25], P < 0.003; 2.49 [1.37-4.50], P < 0.003; and 3.07 [1.70-5.55], P < 0.001, respectively), and MPID (analysis of covariance [ANCOVA] least squares mean [LSM] differences [95% CIs], 25.8 [17.3-34.4], P < 0.003; 20.4 [12.1-28.7], P < 0.003; and 29.3 [20.9-37.6], P < 0.001, respectively). AZD3582 and naproxen did not show any statistically significant differences for the 3 primary variables, except that naproxen was superior to the AZD3582 375-mg dose for the variables time to meaningful pain relief (HR difference, 0.48 [95% CI, 0.29-0.78], P < 0.004) and MPID (difference in ANCOVA LSM, -10.2, [95% CI, -18.2 to -2.2], P < 0.012). The median times to meaningful pain relief were 115 minutes for AZD3582 375 mg, 66 minutes for 750 mg, 85 minutes for 1500 mg, 81 minutes for 2250 mg, and 162 minutes for placebo (P = NS, P = 0.003, P < 0.003, and P < 0.001, respectively). The median time to first rescue medication was 144 minutes for placebo, and <50% of the subjects on any of the AZD3582 doses or naproxen took rescue medication within 8 hours after dosing. In the comparator study, AZD3582 750 mg was superior to placebo in "time to rescue medication (0-24 hours)" (HR [95% CI], 0.4 [0.3-0.6], P < 0.001), "time to confirmed perceptible pain relief" (2.1 [1.1-3.8], P = 0.02), and MPID (11.9 [4.2-19.5], P = 0.002). However, inferiority of AZD3582 to rofecoxib for MPID could not be excluded (tolerance limit of 10 mm; P = NS for noninferiority testing). The median times to confirmed perceptible pain relief were 45 minutes for AZD3582 500 mg, 40 minutes for 750 mg, and 37 minutes for rofecoxib. The median times to first rescue medication were 218 minutes for AZD3582 500 mg, 365 minutes for 750 mg, 635 minutes for rofecoxib, and 90 minutes for placebo. Overall, AZD3582 was well tolerated. However, an effect on orthostatic blood pressure could not be excluded because there seemed to be more subjects with dizziness and orthostatic blood pressure reduction who were administered AZD3582 > or =750 mg. The proportions of patients with vertigo and decreased orthostatic blood pressure each group were as follows: AZD3582 500 mg, 6%; AZD3582 750 mg, 12%; rofecoxib, 3%; and placebo, 5%. CONCLUSIONS: AZD3582 750 mg had similar analgesic efficacy as equimolar doses of naproxen, but noninferiority to rofecoxib was not demonstrated.

Monoclonal antibodies distinguishing alpha and beta forms of calcitonin gene-related peptide.

A panel of 18 monoclonal antibodies was raised to the human calcitonin gene related peptide (CGRP). Of these mabs, seven were specific for alpha CGRP and five for beta CGRP, while the remainder reacted with both alpha and beta CGRP. Nine different epitopes on CGRP were defined with these mabs. In addition, the mabs were tested in various combinations to develop a series of two site assays specific for alpha or for beta CGRP as well as assays able to detect both.

The effect of monoclonal antibodies to calcitonin gene-related peptide (CGRP) on CGRP-induced vasodilatation in pig coronary artery rings.

1. The modification of the vasodilator effect of calcitonin gene-related peptide (CGRP) by a panel of monoclonal antibodies (MAbs), which map to discrete epitopes on the CGRP molecule, was investigated in pig coronary artery rings (PCA). The preparations were pre-constricted with acetylcholine (3 x 10(-7) M) and concentration-response curves to CGRP (2 x 10(-10)-2.56 x 10(-8) M) were obtained in the presence or absence of each MAb. 2. CGRP caused a concentration-dependent relaxation of PCAs which reached a maximum (98.2 +/- 4.8%, n = 25) at 1.28 x 10(-8) M and gave an EC50 of 3.8 +/- 0.8 x 10(-9) M. 3. Two MAbs which map to the N-terminal, CN1 and CRA3, did not affect the CGRP response whilst a third, CRA5, significantly inhibited its effect. 4. The C-terminal MAb, CRA2, did not modify the CGRP response whilst, in contrast, CB3 (C-terminal) potentiated its effect. A similar augmentation of the CGRP-induced vasodilatation was seen in the presence of the middle-region MAb, CRA8. 5. These results suggest that regional specific MAbs can modify the vasodilator effect of CGRP causing either inhibition (CRA5, N-terminal) or potentiation (CB3, C-terminal; CRA8, middle region).

Pharmacological characterization of a receptor for calcitonin gene-related peptide on rat, L6 myocytes.

1 The L6 myocyte cell line expresses high affinity receptors for calcitonin gene-related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation. 2 In intact cells at 37 degrees C, human and rat alpha- and beta-CGRP all activated adenylyl cyclase with EC50s of about 1.5 nM. A number of CGRP analogues containing up to five amino acid substitutions showed similar potencies. In membrane binding studies at 22 degrees C in 1 mM Mg2+, the above all bound to a single site with IC50s of 0.1-0.4 nM. 3 The fragment CGRP(8-37) acted as a competitive antagonist of CGRP stimulation of adenylyl cyclase with a calculated Kd of 5 nM. The Kd determined in membrane binding assays was lower (0.5 nM). 4 The N-terminal extended human alpha-CGRP analogue Tyro-CGRP activated adenylyl cyclase and inhibited [125I]-iodohistidyl-CGRP binding less potently than human alpha-CGRP (EC50 for cyclase = 12 nM, IC50 for binding = 4 nM). 5 The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology.