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Competency-based assessments (CBAs) have gained traction in graduate medical education and inform important learner outcomes through the continuum of medical training. Active participation in new CBAs presents challenges to faculty working in a busy clinical environment. As such, the implementation of new CBAs can be approached with intention to foster acceptance and engagement with new evaluations. This paper describes strategies utilized to implement CBAs among clinician educators during a national assessment pilot. Our methods are grounded in educational, psychological, business, ecological, communication, and information technology theory. Our primary interventions included creating a multilevel vision, engaging a dedicated work group, incorporating quality improvement methodology, and integrating technology to successfully implement the assessments. These practical and effective interventions may also be applied to the implementation of other educational innovations., (Copyright © 2020 Academic Pediatric Association. Published by Elsevier Inc. All rights reserved.)

Neurotrophins and their receptors, the Trks, are differentially expressed among the cell types that make up neuromuscular and other synapses, but the function and directionality of neurotrophin signaling at synapses are poorly understood. Here we demonstrate, via immunostaining, Western blotting, and RT-PCR analyses, that TrkC, the receptor for neurotrophin-3 (NT3), is expressed by mouse perisynaptic and myelinating Schwann cells from birth through adulthood and is unaltered after denervation. Analyses of transgenic mice in which the NT3 coding sequence is replaced by lacZ showed that NT3 is expressed in motor neurons and Schwann cells during perinatal development, but not in adult mice. In muscle, NT3 is expressed by intrafusal muscle fibers within spindles, as has been previously reported. Surprisingly, NT3 is also expressed in extrafusal muscle fibers during perinatal life and in adults. Genetic approaches were used to explore the roles of NT3 and TrkC signaling at neuromuscular synapses. Overexpression of NT3 in muscle fibers during development resulted in an increased number of perisynaptic Schwann cells at neuromuscular synapses, without altering synaptic size, suggesting that muscle-derived NT3 might act as a mitogen or trophic factor for Schwann cells. Conditional deletion of NT3 from motor neurons did not alter the number of Schwann cells or other aspects of neuromuscular synaptic structure, suggesting that motor-neuron-derived NT3 is not required for normal development of perisynaptic Schwann cells or synapses. Together, these results demonstrate that NT3 expression is developmentally regulated in skeletal muscle and may modulate the number of Schwann cells at neuromuscular synapses., ((c) 2007 Wiley-Liss, Inc.)

Hyperthyroid Graves' disease is a common autoimmune disorder mediated by agonistic antibodies to the TSH receptor, termed thyroid stimulating antibodies (TSAbs). Recently members of the TNF superfamily, B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), have been identified along with their receptors, B cell maturation antigen and transmembrane activator and calcium-modulator and cyclophilin ligand interactor, and the BAFF-specific receptor. BAFF is a fundamental B cell survival/maturation factor, and both BAFF and APRIL have been implicated in antibody production. We investigated the effect of interfering with BAFF- and APRIL-mediated signals in an induced model of Graves' disease by blockade of these factors using soluble decoy receptors. In a therapeutic setting in mice with established hyperthyroidism, we show that blockade of BAFF or BAFF+APRIL with BAFF-specific receptor-Fc and B cell maturation antigen-Fc, respectively, leads to significant reductions in the induced hyperthyroidism. This was supported by a parallel pattern of declining TSAbs in the responding animals. Histopathological analysis of splenic sections from treated animals revealed marked reductions in the B cell follicle regions, but staining with anti-CD138 revealed the persistence of plasma cells. Thus, the reductions in TSAbs in the treated animals were not related to overall plasma cell numbers in the secondary lymphoid organs. Our results are the first to demonstrate attenuation of established hyperthyroidism by therapeutic intervention aimed at autoreactive B cells and indicate that both BAFF and APRIL appear to play important roles in the development and survival of the autoantibody producing cells in this model.

PEGylation of IFN-alpha has been used successfully to improve the pharmacokinetic properties and efficacy of the drug. To prepare a PEGylated form of human interferon-beta-1a (IFN-beta-1a) suitable for testing in vivo, we have synthesized 20 kDa mPEG-O-2-methylpropionaldehyde and used it to modify the N-terminal alpha-amino group of the cytokine. The PEGylated protein retained approximately 50% of the activity of the unmodified protein and had significantly improved pharmacokinetic properties following intravenous administration in rats. The clearance and volume of distribution at steady state were reduced approximately 30-fold and approximately 4-fold, respectively, resulting in a significant increase in systemic exposure as determined by the area under the curve. The elimination half-life of the PEGylated protein was approximately 13-fold greater than for the unmodified protein. The unmodified and PEGylated proteins were tested for their ability to inhibit the formation of radially oriented blood vessels entering the periphery of human SK-MEL-1 melanoma tumors in athymic nude homozygous (nu/nu) mice. In a single dose comparison study, administration of 1 x 10(6) units of unmodified IFN-beta-1a resulted in a 29% reduction in vessel number, while 1 x 10(6) units of PEGylated IFN-beta-1a resulted in a 58% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle (control)-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. In a multiple versus single dose comparison study, daily administration of 1 x 10(6) units of unmodified IFN-beta-1a for 9 days resulted in a 51% reduction in vessel number, while a single dose of 1 x 10(6) units of the PEGylated protein resulted in a 66% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. Therefore, the improved pharmacokinetic properties of the modified protein translated into improved efficacy. Since unmodified IFN-beta is used for the treatment of multiple sclerosis and hepatitis C virus infection, a PEGylated form of the protein such as 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-beta-1a may serve as a useful adjunct for the treatment of these diseases. In addition, the antiangiogenic effects of PEGylated IFN-beta-1a may be harnessed for the treatment of certain cancers, either as a sole agent or in combination with other antitumor drugs.

We report that B cell-activating factor of the tumor necrosis factor (TNF) family (BAFF) is expressed in the normal human brain at approximately 10% of that in lymphatic tissues (tonsils and adenoids) and is produced by astrocytes. BAFF was regularly detected by enzyme-linked immunosorbent assay in brain tissue lysates and in normal spinal fluid, and in astrocytes by double fluorescence microscopy. Cultured human astrocytes secreted functionally active BAFF after stimulation with interferon-gamma and TNF-alpha via a furin-like protease-dependent pathway. BAFF secretion per cell was manifold higher in activated astrocytes than in monocytes and macrophages. We studied brain lesions with B cell components, and found that in multiple sclerosis plaques, BAFF expression was strongly up-regulated to levels observed in lymphatic tissues. BAFF was localized in astrocytes close to BAFF-R-expressing immune cells. BAFF receptors were strongly expressed in situ in primary central nervous system (CNS) lymphomas. This paper identifies astrocytes as a nonimmune source of BAFF. CNS-produced BAFF may support B cell survival in inflammatory diseases and primary B cell lymphoma.

Blockade of the CD154-CD40 co-stimulatory pathway with anti-CD154 mAbs has shown impressive efficacy in models of autoimmunity and allotransplantation. Clinical benefit was also demonstrated in systemic lupus erythematosus (SLE) and idiopathic thrombocytopenia patients with the humanized anti-CD154 mAb, 5C8 (hu5C8). However, thromboembolic complications that occurred during the course of the hu5C8 clinical trials have proven to be a major setback to the field and safe alternative therapeutics targeting the CD154-CD40 pathway are of great interest. Recently, effector mechanisms have been shown to play a part in anti-CD154 mAb-induced transplant acceptance in murine models, while this issue remains unresolved for humoral-mediated models. Herein, aglycosyl anti-CD154 mAbs with reduced binding to FcgammaR and complement were used as a novel means to test the role of effector mechanisms in non-human primate and murine models not amenable to gene knockout technology. While aglycosyl hu5C8 mAb was relatively ineffective in rhesus renal and islet allotransplantation, it inhibited primary and secondary humoral responses to a protein immunogen in cynomolgus monkeys. Moreover, an aglycosyl, chimeric MR1 mAb (muMR1) prolonged survival and inhibited pathogenic auto-antibody production in a murine model of SLE. Thus, the mechanisms required for efficacy of anti-CD154 mAbs depend on the nature of the immune challenge.

It has been demonstrated that anti-CD154 mAb treatment effectively inhibits the development of experimental autoimmune encephalomyelitis (EAE). However, although it appears to prevent the induction of Th1 cells and reactivation of encephalitogenic T cells within the CNS, little information is available regarding the involvement of alternative mechanisms, nor has the contribution of Fc effector mechanisms in this context been addressed. By contrast, efficacy of anti-CD154 mAbs in models of allotransplantation has been reported to involve long-term unresponsiveness, potentially via activation of T regulatory cells, and recently was reported to depend on Fc-dependent functions, such as activated T cell depletion through FcgammaR or complement. In this study we demonstrate that anti-CD154 mAb treatment inhibits EAE development in SJL mice without apparent long-term unresponsiveness or active suppression of disease. To address whether the mechanism of inhibition of EAE by anti-CD154 mAb depends on its Fc effector interactions, we compared an anti-CD154 mAb with its aglycosyl counterpart with severely impaired FcgammaR binding and reduced complement binding activity with regard to their ability to inhibit clinical signs of EAE and report that both forms of the Ab are similarly protective. This observation was largely confirmed by the extent of leukocyte infiltration of the CNS; however, mice treated with the aglycosyl form may display slightly more proteolipid protein 139-151-specific immune reactivity. It is concluded that FcR interactions do not play a major role in the protective effect of anti-CD154 mAb in the context of EAE, though they may contribute to the full abrogation of peripheral peptide-specific lymphocyte responses.

We report a random disruption in the mouse genome that resulted in lethal paralysis in homozygous newborns. The disruption blocked expression of neurobeachin, a protein containing a BEACH (beige and Chediak-Higashi) domain implicated in synaptic vesicle trafficking and an AKAP (A-kinase anchor protein) domain linked to localization of cAMP-dependent protein kinase activity. nbea-null mice demonstrated a complete block of evoked synaptic transmission at neuromuscular junctions, whereas nerve conduction, synaptic structure, and spontaneous synaptic vesicle release were completely normal. These findings support an essential role for neurobeachin in evoked neurotransmitter release at neuromuscular junctions and suggest that it plays an important role in synaptic transmission.

Peripheral autoantigen binding B cells are poorly competitive with naive B cells for survival and undergo rapid cell death. However, in monoclonal Ig-transgenic mice lacking competitor B cells, autoantigen binding B cells can survive for extended periods. The basis for competitive elimination of autoantigen binding B cells has been unknown. Here we demonstrate that autoantigen binding B cells have increased dependence on BAFF for survival. In monoclonal Ig-transgenic mice, each autoantigen binding B cell receives elevated amounts of BAFF, exhibiting increased levels of NFkappaB p52 and of the prosurvival kinase Pim2. When placed in a diverse B cell compartment, BAFF receptor engagement and signaling are reduced and the autoantigen binding cells are unable to protect themselves from Bim and possibly other death-promoting factors induced by chronic BCR signaling. These findings indicate that under conditions where BAFF levels are elevated, autoantigen-engaged cells will be rescued from rapid competitive elimination, predisposing to the development of autoimmune disease.

To identify potential new clinical uses and routes of administration for human interferon-beta-1a (IFN-beta-1a), we have developed an expression and purification procedure for the preparation of highly purified rat interferon-beta (IFN-beta) suitable for testing in rat models of human disease. An expression vector containing the rat IFN-beta signal sequence and structural gene was constructed and transfected into Chinese hamster ovary (CHO) cells. The protein was purified from CHO cell conditioned medium and purified to > 99.5% purity using standard chromatographic techniques. Analytical characterization indicated that the protein was a heavily glycosylated monomeric protein, with two of the four predicted N-glycosylation sites occupied. Analysis of the attached oligosaccharides showed them to be a complex mixture of bi-antennary, tri-antennary, and tetra-antennary structures with a predominance of sialylated tri-antennary and tetra-antennary structures. Peptide mapping, N-terminal sequencing, and mass spectrometry confirmed the identity and integrity of the purified protein. The purified protein had a specific activity of 2.1x10(8)U/mg when assayed on rat RATEC cells, which is similar in magnitude to the potencies observed for murine IFN-beta and human IFN-beta-1a assayed on murine and human cells, respectively. We also prepared an N-terminally PEGylated form of rat IFN-beta in which a 20 kDa methoxy polyethylene glycol (PEG)-propionaldehyde was attached to the N-terminal alpha-amino group of Ile-1. The PEGylated protein, which retained essentially full in vitro antiviral activity, had improved pharmacokinetic parameters in rats as compared to the unmodified protein. Both the unmodified and PEGylated forms of rat IFN-beta will be useful for testing in rat models of human disease.

The cell surface co-stimulatory protein CD154 (CD40L) is a target for monoclonal antibody (mAb) inhibitors of T-cell mediated immune diseases. This protein, like most other members of the TNF ligand family, forms homotrimeric complexes on the cell surface and in solution, with a three-fold axis of symmetry. We find that several different anti-CD154 monoclonal antibodies form distinctive complexes with soluble CD154. These soluble complexes have been analyzed using size exclusion chromatography, static and dynamic light scattering, and electron microscopy and shown to consist of caged structures of various geometries. The cell surface complexes have been analyzed by confocal microscopy and, depending on the mAb, remain as small, separate complexes or form large aggregates. The formation of these complexes in solution is likely to have an impact on measures of affinity, while the cell surface complexes could affect binding potency and provoke other biological effects.

Islet transplantation for the treatment of autoimmune diabetes is more difficult because of the additional barrier presented by the autoimmunity. We tested the ability of hamster anti-rat CD154 to prevent recurrence of diabetes in renal subcapsular islet isografts in DR-BB (RT1uu) rats with established autoimmune diabetes. Experimental animals with established diabetes received intravenous injections of 15 mg/kg anti-CD154 on a specified schedule starting 2 days before renal subcapsular transplantation of an islet isograft. Control animals received either saline or hamster IgG. Plasma glucose levels >250 mg/dl over 3 days were used to indicate the recurrence of diabetes. Rats that received saline (n = 5) or control antibody (n = 3) had a recurrence of diabetes 6-11 days after transplantation. Histological examination of islet isografts from these rats showed complete destruction of the insulin-producing portion of the isograft with residual cells positive for glucagon. Recipient rats that received anti-CD154 at the 15-mg/kg dosage (n = 6) did not have a recurrence of diabetes for 308-461 days after transplantation. Islet isografts removed from the rats showed low levels of insulin immunoreactivity, high levels of insulin mRNA, and focal infiltration with lymphocytes but no evidence of islet destruction. Mean peak antibody concentration was 266 microg/ml and returned to undetectable levels by 67-88 days after transplantation. Rats that received anti-CD154 starting at 4-7 days after transplantation had a recurrence of diabetes within 11 days of the isotransplantation. Therefore, anti-CD154 as the sole immunomodulator prevented the recurrence of diabetes in islet isografts in rats with established autoimmune diabetes. This suggests that CD40/CD154 blockade is effective in preventing the insulitis or the effector phase of autoimmune diabetes.

Background: Treatment of patients with secondary pulmonary hypertension has been unsatisfactory., Objective: To describe exercise capacity, functional class, and hemodynamic variables after long-term intravenous infusion of prostacyclin in patients with secondary pulmonary hypertension., Design: Case series., Setting: Academic referral center., Patients: 33 patients with secondary, precapillary pulmonary hypertension (New York Heart Association class III or IV)., Intervention: Continuous intravenous prostacyclin administered by portable infusion pump on a compassionate-use basis., Measurements: Functional class, treadmill time, and hemodynamic variables., Results: Patients were followed for an average of 12.7 +/- 5.6 months. Exercise tolerance and New York Heart Association class improved in each patient. The duration of treadmill exercise increased from 186 seconds to 491 seconds, an increase of 305 seconds (95% CI, 194 to 417 seconds; P < 0.001). Mean pulmonary artery pressure decreased from 60 mm Hg to 46 mm Hg, a decrease of 14 mm Hg (CI, 9 to 19 mm Hg; P < 0.001). Cardiac output increased from 3.90 L/min to 6.30 L/min, an increase of 2.40 L/min (CI, 1.56 to 3.25 L/min; P < 0.001). The pulmonary vascular resistance decreased from 1143 dynes x s/cm5 to 575 dynes x s/cm5, a decrease of 567 dynes x s/cm5 (CI, 407 to 727 dynes x s/cm5; P < 0.001). Patients with collagen vascular disease, congenital heart disease, and portopulmonary hypertension were analyzed with other patients and separately. All groups had a statistically significant reduction in mean pulmonary artery pressure and a statistically significant increase in cardiac output., Conclusion: Intravenous prostacyclin may be effective in the treatment of patients with certain types of secondary pulmonary hypertension.

The pharmacokinetics and pharmacodynamics (PK/PD) of chimeric (Ch5c8) and humanized (Hu5c8) 5c8, a monoclonal antibody that binds CD154 (CD40 ligand), thus blocking the interaction between CD40 and CD154, were investigated in cynomolgus monkeys. Single-dose groups (n = 3 animals per dose) received saline, 0.2, 1, 5 or 20 mg/kg i.v. doses of Hu5c8. The repeat-dose groups (n = 4 animals) received 0 or 5 mg/kg i.v. doses of Ch5c8 or Hu5c8 on days 1, 2, 3, 5, 7 and 9. The single-dose PK parameters showed dose proportionality, with a terminal half-life of 300 h, a volume of distribution at steady state of 73 ml/kg and clearance of 0.2 ml.h-1.kg-1. The repeat-dose regimen produced a longer terminal half-life (500 h) and lower clearance (0.13 ml.h-1.kg-1) than in the single-dose groups. The antibody titer to tetanus toxoid (ATT) challenge served as the immunodynamic marker. The primary ATT response consisted of a latent phase of approximately 10 days, during which the immune system was processing antigen but not yet producing antibody, a rise to an antibody maximum titer at approximately 18 days and a decline toward baseline by approximately 40 days in controls. The 5c8 produced a log(dose)-proportional reduction in the area under the curve of ATT. An indirect PK/PD model based on the kinetics of tetanus toxoid exposure and inhibition of ATT production in relation to 5c8 concentrations was developed. A median inhibitory concentration of 0.84 microg/ml and a efficacy of 0.84 reflected marked inhibition of ATT response by 5c8. The model provides quantitation of reduced ATT responses after 5c8 and was applicable to primary and secondary immune responses and to both single-dose and multiple-dose treatments. The monoclonal antibody 5c8 blocks the CD40 and CD154 interaction, producing consistent and substantive reduction in antibody formation after administration of tetanus toxoid, which can be characterized with PK/PD modeling. It is anticipated that 5c8 may have utility in the treatment of antibody-mediated autoimmune disease.

Hyperthermia has been found to be a useful modality for cancer therapy. In this report, a biocompatible, ferrimagnetic glass-ceramic capable of inducing localized hyperthermia by hysteresis heating upon exposure to an alternating magnetic field is presented. When the glass-ceramic was placed in the region of a subcutaneously transplanted, weakly antigenic breast carcinoma and subjected to the magnetic field, sufficient temperature rise was obtained to cause significant (approximately 50%) tumor regrowth delay and a 12% permanent control. The data demonstrate that glass-ceramic-mediated hysteresis heating may be a useful therapeutic approach in the treatment of cancer which offers the advantage of producing a highly localized and predictable tumor volume hyperthermia.

Bovine spermatozoa were separated into different density subpopulations utilizing water insoluble hydrocarbon and silicone gels of defined specific gravity. Sperm density profiles were generated for 13 bulls. The separations were found to be repeatable and characteristic of the bull examined. Considerable density variation among animals was demonstrated. Analysis of the separated spermatozoa, before and after freezing, demonstrated that good motility and acrosomal integrity of spermatozoa were maintained. When the least dense fraction of spermatozoa was used for insemination, conception rates were similar to those obtained routinely by artificial insemination with unfractionated spermatozoa. Therefore, this system may be useful in separating spermatozoa of various densities and for removing extraneous matter from semen. However, the sex ratio, among 51 60-day-old fetuses recovered from heifers inseminated with the lowest density fraction of spermatozoa, was 26 males:25 females.