Use of poxvirus display to select antibodies specific for complex membrane antigens.

Antibody discovery against complex antigens is limited by the availability of a reproducible pure source of concentrated properly folded antigen. We have developed a technology to enable direct incorporation of membrane proteins such as GPCRs and into the membrane of poxvirus. The protein of interest is correctly folded and expressed in the cell-derived viral membrane and does not require any detergents or refolding before downstream use. The poxvirus is selective in which proteins are incorporated into the viral membrane, making the antigen poxvirus an antigenically cleaner target for in vitro panning. Antigen-expressing virus can be readily purified at scale and used for antibody selection using any in vitro display platform.

A Vaccinia-based system for directed evolution of GPCRs in mammalian cells.

Directed evolution in bacterial or yeast display systems has been successfully used to improve stability and expression of G protein-coupled receptors for structural and biophysical studies. Yet, several receptors cannot be tackled in microbial systems due to their complex molecular composition or unfavorable ligand properties. Here, we report an approach to evolve G protein-coupled receptors in mammalian cells. To achieve clonality and uniform expression, we develop a viral transduction system based on Vaccinia virus. By rational design of synthetic DNA libraries, we first evolve neurotensin receptor 1 for high stability and expression. Second, we demonstrate that receptors with complex molecular architectures and large ligands, such as the parathyroid hormone 1 receptor, can be readily evolved. Importantly, functional receptor properties can now be evolved in the presence of the mammalian signaling environment, resulting in receptor variants exhibiting increased allosteric coupling between the ligand binding site and the G protein interface. Our approach thus provides insights into the intricate molecular interplay required for GPCR activation.

Generation and preclinical characterization of an antibody specific for SEMA4D.

Semaphorin 4D (SEMA4D or CD100) is a member of the semaphorin family of proteins and an important mediator of the movement and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. Blocking the binding of SEMA4D to its receptors can result in physiologic changes that may have implications in cancer, autoimmune, and neurological disease. To study the effects of blocking SEMA4D, we generated, in SEMA4D-deficient mice, a panel of SEMA4D-specific hybridomas that react with murine, primate, and human SEMA4D. Utilizing the complementarity-determining regions from one of these hybridomas (mAb 67-2), we generated VX15/2503, a humanized IgG4 monoclonal antibody that is currently in clinical development for the potential treatment of various malignancies and neurodegenerative disorders, including multiple sclerosis and Huntington's disease. This work describes the generation and characterization of VX15/2503, including in vitro functional testing, epitope mapping, and an in vivo demonstration of efficacy in an animal model of rheumatoid arthritis.

Influenza enhances caspase-1 in bronchial epithelial cells from asthmatic volunteers and is associated with pathogenesis.

BACKGROUND: The leading cause of asthma exacerbation is respiratory viral infection. Innate antiviral defense pathways are altered in the asthmatic epithelium, yet involvement of inflammasome signaling in virus-induced asthma exacerbation is not known. OBJECTIVE: This study compared influenza-induced activation of inflammasome and innate immune signaling in human bronchial epithelial cells from volunteers with and without asthma and investigated the role of caspase-1 in epithelial cell antiviral defense. METHODS: Differentiated primary human bronchial epithelial cells from volunteers with and without asthma were infected with influenza A virus. An inflammasome-specific quantitative real-time polymerase chain reaction array was used to compare baseline and influenza-induced gene expression profiles. Cytokine secretion, innate immune gene expression, and viral replication were compared between human bronchial epithelial cells from volunteers with and without asthma. Immunofluorescence microscopy was used to evaluate caspase-1 and PYCARD colocalization. Tracheal epithelial cells from caspase-1-deficient or wild-type mice were infected with influenza and assessed for antiviral gene expression and viral replication. RESULTS: Human bronchial epithelial cells from asthmatic volunteers had altered influenza-induced expression of inflammasome-related and innate immune signaling components, which correlated with enhanced production of IL-1ß, IL-6, and TNF-α. Specifically, influenza-induced caspase-1 expression was enhanced and localization differed in human bronchial epithelial cells from asthmatic volunteers compared to volunteers without asthma. Influenza-infected tracheal epithelial cells from caspase-1-deficient mice had reduced expression of antiviral genes and viral replication. CONCLUSION: Caspase-1 plays an important role in the airway epithelial cell response to influenza infection, which is enhanced in asthmatic volunteers, and may contribute to the enhanced influenza-related pathogenesis observed in vivo.

Cerium dioxide nanoparticles can interfere with the associated cellular mechanistic response to diesel exhaust exposure.

The aim of this study was to compare the biological response of a sophisticated in vitro 3D co-culture model of the epithelial airway barrier to a co-exposure of CeO(2) NPs and diesel exhaust using a realistic air-liquid exposure system. Independent of the individual effects of either diesel exhaust or CeO(2) NPs investigation observed that a combined exposure of CeO(2) NPs and diesel exhaust did not cause a significant cytotoxic effect or alter cellular morphology after exposure to diesel exhaust for 2h at 20µg/ml (low dose) or for 6h at 60µg/ml (high dose), and a subsequent 6h exposure to an aerosolized solution of CeO(2) NPs at the same doses. A significant loss in the reduced intracellular glutathione level was recorded, although a significant increase in the oxidative marker HMOX-1 was found after exposure to a low and high dose respectively. Both the gene expression and protein release of tumour necrosis factor-α were significantly elevated after a high dose exposure only. In conclusion, CeO(2) NPs, in combination with diesel exhaust, can significantly interfere with the cell machinery, indicating a specific, potentially adverse role of CeO(2) NPs in regards to the biological response of diesel exhaust exposure.

Toxic effects of brake wear particles on epithelial lung cells in vitro.

BACKGROUND: Fine particulate matter originating from traffic correlates with increased morbidity and mortality. An important source of traffic particles is brake wear of cars which contributes up to 20% of the total traffic emissions. The aim of this study was to evaluate potential toxicological effects of human epithelial lung cells exposed to freshly generated brake wear particles. RESULTS: An exposure box was mounted around a car's braking system. Lung cells cultured at the air-liquid interface were then exposed to particles emitted from two typical braking behaviours ("full stop" and "normal deceleration"). The particle size distribution as well as the brake emission components like metals and carbons was measured on-line, and the particles deposited on grids for transmission electron microscopy were counted. The tight junction arrangement was observed by laser scanning microscopy. Cellular responses were assessed by measurement of lactate dehydrogenase (cytotoxicity), by investigating the production of reactive oxidative species and the release of the pro-inflammatory mediator interleukin-8. The tight junction protein occludin density decreased significantly (p < 0.05) with increasing concentrations of metals on the particles (iron, copper and manganese, which were all strongly correlated with each other). Occludin was also negatively correlated with the intensity of reactive oxidative species. The concentrations of interleukin-8 were significantly correlated with increasing organic carbon concentrations. No correlation was observed between occludin and interleukin-8, nor between reactive oxidative species and interleukin-8. CONCLUSION: These findings suggest that the metals on brake wear particles damage tight junctions with a mechanism involving oxidative stress. Brake wear particles also increase pro-inflammatory responses. However, this might be due to another mechanism than via oxidative stress.

Management of chronic posttraumatic headache: a multidisciplinary approach.

Although the definitions and possible treatments of posttraumatic headache have been discussed in the literature, treatment of this condition is not standardized. In addition, though various reviews and case studies have supported the use of spinal manipulation in the treatment of tension-type headaches and migraines, few describe the use of osteopathic manipulative treatment for patients with posttraumatic headache. In describing a 38-year-old woman with posttraumatic headache, we illustrate a multidisciplinary approach-including the use of osteopathic principles and practice-to treating patients with posttraumatic headache.

A newly developed in vitro model of the human epithelial airway barrier to study the toxic potential of nanoparticles.

The potential health effects of inhaled engineered nanoparticles are almost unknown. To avoid and replace toxicity studies with animals, a triple cell co-culture system composed of epithelial cells, macrophages and dendritic cells was established, which simulates the most important barrier functions of the epithelial airway. Using this model, the toxic potential of titanium dioxide was assessed by measuring the production of reactive oxygen species and the release of tumour necrosis factor alpha. The intracellular localisation of titanium dioxide nanoparticles was analyzed by energy filtering transmission electron microscopy. Titanium dioxide nanoparticles were detected as single particles without membranes and in membrane-bound agglomerates. Cells incubated with titanium dioxide particles showed an elevated production of reactive oxygen species but no increase of the release of tumour necrosis factor alpha. Our in vitro model of the epithelial airway barrier offers a valuable tool to study the interaction of particles with lung cells at a nanostructural level and to investigate the toxic potential of nanoparticles.

Rizatriptan efficacy in ICHD-II pure menstrual migraine and menstrually related migraine.

OBJECTIVE: To examine the efficacy of rizatriptan for the treatment of pure menstrual migraine (PMM). BACKGROUND: In 2004, the International Headache Society proposed new research criteria for menstrual migraine (International Classification of Headache Disorders [ICHD-II]). Two subtypes were defined: PMM, in which attacks occur exclusively with menstruation, and menstrually related migraine (MRM), in which attacks may also occur at other times of the cycle. METHODS: The 2 protocols (MM1 and MM2) were identical randomized, double-blind studies. Adult patients with ICHD-II menstrual migraine were assigned to either rizatriptan 10-mg tablet or placebo (2:1). Patients were to treat a single menstrual migraine attack of moderate or severe pain intensity. This prospectively planned substudy pooled data from patients with a diagnosis of PMM from both studies. The primary substudy endpoint was 2-hour pain relief. Efficacy data were summarized for patients with a diagnosis of MRM. RESULTS: Of 707 (MM1: 357, MM2: 350) patients treated in the study, 146 patients (MM1: 81, MM2: 65) had a diagnosis of PMM. The percentage of patients reporting 2-hour pain relief was significantly greater for rizatriptan than for placebo for both PMM (73% vs 50%, P = .006) and MRM subgroups (71% vs 52%, P < .001). Most other efficacy endpoints favored rizatriptan compared with placebo in patients with either PMM or MRM. CONCLUSION: Rizatriptan 10 mg was superior to placebo for the treatment of PMM, as measured by 2-hour pain relief. Rizatriptan was also effective for the treatment of MRM and for relief of migraine-associated symptoms for both headache subtypes.

Diagnosing and managing migraine headache.

Headache is one of the chief complaints among patients visiting primary care physicians. Diagnosis begins with exclusion of secondary causes for headache. More than 90% of patients will have a primary-type headache, so diagnosis can often be completed without further testing. Although tension-type headaches are the most common kind of headache, patients with this type of headache rarely seek treatment unless occurrence is daily. Migraine, which affects more than 30 million people in the United States, is the most common headache diagnosis for which patients seek treatment. Migraine is a chronic, often inherited condition involving brain hypersensitivity and a lowered threshold for trigeminal-vascular activation. Intermittent debilitating attacks are characterized by autonomic, gastrointestinal, and neurologic symptoms. Migraine results in a marked decrease in a patient's quality of life, as measured by physical, mental, and social health-related instruments. Accurate assessment of a patient's disability will guide physicians in prescribing appropriate modes of therapy. However, migraine remains underdiagnosed, and patients with migraine remain undertreated. A comprehensive treatment approach to migraine may include nonpharmacologic measures, as well as abortive and prophylactic medications. Informing patients about realistic treatment expectations, possible delayed efficacy of medications, and avoidance of caffeine and overuse of medications is critical for successful outcomes. Management of migraine is a dynamic process, because headaches evolve over time and medication tachyphylaxis may occur, necessitating changes in therapy. Pathologic findings in the neck constitute an accepted etiology or precipitant for headache. Osteopathic manipulative treatment may reduce pain input into the trigeminal nucleus caudalis, favorably altering neuromuscular-autonomic regulatory mechanisms to reduce discomfort from headache.

Headache in medical education: medical schools, neurology and family practice residencies.

OBJECTIVE: To explore the extent of headache education received by medical students and residents. BACKGROUND: Headache is a common, often severe, and sometimes disabling problem. However, 49% of sufferers do not seek professional treatment-of those who do, only 28% are very satisfied. One possible reason is limited education of physicians about headache. METHODS: Surveys were sent to all allopathic and osteopathic medical schools, 200 family medicine residencies, and all 126 neurology residencies. Information requested included the amount and perceived adequacy of headache education and any plans to increase headache education. RESULTS: Response rates were 35% to 40%. Medical school lecture hours ranged from 0 (4%) to >5 (24%) with 92% having no plans for an increase in headache education. Family Medicine residency lecture hours ranged from 1--3 (30%) to >5 (34%) and case presentations from 1--5 (23%) to >5 (41%), with 88% of program directors having no plans for increase. Neurology residency lecture hours ranged from 1--3 (11%) to >5 (64%) and case presentations from 1--5 (23%) to>0 (57%), with 80% having no plans for increase. CONCLUSION: Undergraduate medical education in headache is limited. Despite medical schools perceiving their training as adequate, both neurology and family practice residency program directors believe entering residents are inadequately prepared in headache upon entering the program.

Efficacy and tolerability of oral zolmitriptan in menstrually associated migraine: a randomized, prospective, parallel-group, double-blind, placebo-controlled study.

BACKGROUND: Approximately 60% of female migraineurs report experiencing migraine in association with menstruation, while 7% to 25% experience attacks almost exclusively with menstruation. OBJECTIVE: To examine the efficacy and tolerability of oral zolmitriptan in the acute treatment of menstrually associated migraine. In this study, menstrually associated migraine was defined as migraine that consistently occurred from 72 hours before to 5 days after onset of menses. Methods.-Participants were women with regular menstrual cycles, aged 18 to 55 years, who had experienced migraine with at least two thirds of prior menstrual cycles. Subjects were randomized to treat one attack per menstrual cycle for 3 months with either zolmitriptan or placebo. Treatment was intensity based: mild migraines were treated with half of a 2.5-mg zolmitriptan tablet, moderate migraines were treated with zolmitriptan 2.5 mg, and severe migraines were treated with 5 mg (two 2.5-mg tablets) of zolmitriptan, or placebo. RESULTS: Of the 579 women enrolled in the study, 260 were treated with zolmitriptan and 251 were assigned placebo. Twelve hundred thirty-two attacks were treated, and a 2-hour headache response was achieved in 48% of zolmitriptan-treated attacks as compared with 27% of placebo-assigned attacks (P <.0001). Zolmitriptan was superior to placebo in achieving a headache response as early as 30 minutes (18% versus 14%, P=.03) and at 1 hour (33% versus 23%, P <.001). Drug-related adverse events were reported in 16% of subjects receiving zolmitriptan and 9% of subjects receiving placebo. CONCLUSION: Oral zolmitriptan exhibits efficacy and good tolerability in the treatment of menstrually associated migraine. Improvement over placebo was observed as early as 30 minutes following treatment.

Managing intractable migraine with naratriptan.

BACKGROUND: Intractable migraine presents a significant treatment challenge to both patient and physician. Most attacks are treatable or self-limiting, but occasionally they may continue for extended periods regardless of treatment. OBJECTIVE: To determine the efficacy of naratriptan 2.5 mg twice daily for the treatment of intractable migraine. METHODS: We reviewed 24 patients treated with naratriptan twice daily for an intractable migraine attack. Patients were permitted to take prophylactic medication if such treatment had been effective in the past. RESULTS: Nineteen patients (79%) improved. Twelve patients showed excellent response with cessation of pain and associated symptoms, 7 patients partially responded with lessening of pain and cessation of associated symptoms, and 5 patients were nonresponsive. CONCLUSION: Short-term daily administration of naratriptan may be effective in terminating status migrainosus.

Tension-type, the forgotten headache. How to recognize this common but undertreated condition.

Tension-type headache is the most prevalent headache type, affecting most women and men at some time in their lives, and can impair job productivity and interfere with family and social time. However, despite its impact, tension-type headache is also the most misunderstood headache type. Because of these misperceptions, many affected people are reluctant to seek medical treatment. In this article, Dr Mueller discusses possible triggers of tension-type headache, appropriate evaluation, and management with pharmacologic, nonpharmacologic, and alternative therapies.

Divalproex sodium in the treatment of migraine and cluster headaches.

The discovery of a new class of effective migraine-abortive medications, the triptans, has sparked a new interest in the study of vascular headache. Over the past few years, the Food and Drug Administration (FDA) has approved six new abortive pharmacologic therapies, with several others in various stages of clinical trials. Unfortunately, concurrent pharmacologic changes in headache prophylaxis have not kept pace with their abortive counterparts. However, divalproex sodium (Depakote), which is approved by the FDA as a migraine prophylactic agent, is the first in the anticonvulsant class of medication for migraine headache and has expanded the options in headache treatment. The objective of this retrospective multicenter study of 284 patients with migraine or cluster headaches was to examine the clinical efficacy and safety of divalproex sodium as prophylaxis in monotherapy and in polytherapy. Sixty-one percent of migraineurs and 73% of cluster patients noted a decrease in pain with divalproex sodium and continued that therapy for more than 3 months. Reported negative side effects included weight gain, nausea, somnolence, tremor, alopecia, dysequilibrium, and rash. However, only 14% of subjects discontinued therapy due to these side effects. Overall, divalproex sodium was found to be an effective and generally well-tolerated prophylactic treatment option as monotherapy or in polytherapy for migraine and cluster headache.

Smoking in women.

Smoking has numerous increased health risks for women, including the risks for cancer, cardiovascular disease, chronic obstructive pulmonary disease, gastric and duodenal ulcers, reduced fertility, ovulatory dysfunction, ectopic pregnancy, spontaneous abortion, sudden infant death, and earlier menopause. Such health risks, smoking cessation therapy, and unique obstacles to smoking cessation in women are the focus of this article.