Identification of alpha-gal sensitivity in patients with a diagnosis of idiopathic anaphylaxis.

IgE antibodies (Ab) specific to galactose-α-1,3-galactose (alpha-gal) are responsible for a delayed form of anaphylaxis that occurs 3-6 hours after red meat ingestion. In a unique prospective study of seventy participants referred with a diagnosis of idiopathic anaphylaxis (IA), six (9%) were found to have IgE to alpha-gal. Upon institution of a diet free of red meat, all patients had no further episodes of anaphylaxis. Two of these individuals had indolent systemic mastocytosis (ISM). Those with ISM had more severe clinical reactions but lower specific IgE to alpha-gal and higher serum tryptase levels, reflective of the mast cell burden. The identification of alpha-gal syndrome in patients with IA supports the need for routine screening for this sensitivity as a cause of anaphylaxis, where reactions to alpha-gal are delayed and thus may be overlooked.

Inhibition of autophagy in EBV-positive Burkitt's lymphoma cells enhances EBV lytic genes expression and replication.

Autophagy, an important degradation system involved in maintaining cellular homeostasis, serves also to eliminate pathogens and process their fragments for presentation to the immune system. Several viruses have been shown to interact with the host autophagic machinery to suppress or make use of this cellular catabolic pathway to enhance their survival and replication. Epstein Barr virus (EBV) is a γ-herpes virus associated with a number of malignancies of epithelial and lymphoid origin in which establishes a predominantly latent infection. Latent EBV can periodically reactivate to produce infectious particles that allow the virus to spread and can lead to the death of the infected cell. In this study, we analyzed the relationship between autophagy and EBV reactivation in Burkitt's lymphoma cells. By monitoring autophagy markers and EBV lytic genes expression, we demonstrate that autophagy is enhanced in the early phases of EBV lytic activation but decreases thereafter concomitantly with increased levels of EBV lytic proteins. In a cell line defective for late antigens expression, we found an inverse correlation between EBV early antigens expression and autophagosomes formation, suggesting that early after activation, the virus is able to suppress autophagy. We report here for the first time that inhibition of autophagy by Bafilomycin A1 or shRNA knockdown of Beclin1 gene, highly incremented EBV lytic genes expression as well as intracellular viral DNA and viral progeny yield. Taken together, these findings indicate that EBV activation induces the autophagic response, which is soon inhibited by the expression of EBV early lytic products. Moreover, our findings open the possibility that pharmacological inhibitors of autophagy may be used to enhance oncolytic viral therapy of EBV-related lymphomas.

Anaphylaxis and cardiovascular disease: therapeutic dilemmas.

Cardiovascular disease (CVD) increases the risk of severe or fatal anaphylaxis, and some medications for CVD treatment can exacerbate anaphylaxis. The aim of this article is to review the effect of anaphylaxis on the heart, the potential impact of medications for CVD on anaphylaxis and anaphylaxis treatment, and the cardiovascular effects of epinephrine. The therapeutic dilemmas arising from these issues are also discussed and management strategies proposed. PubMed searches were performed for the years 1990-2014 inclusive, using terms such as angiotensin-converting enzyme (ACE) inhibitors, adrenaline, allergic myocardial infarction, anaphylaxis, angiotensin-receptor blockers (ARBs), beta-adrenergic blockers, epinephrine, and Kounis syndrome. Literature analysis indicated that: cardiac mast cells are key constituents of atherosclerotic plaques; mast cell mediators play an important role in acute coronary syndrome (ACS); patients with CVD are at increased risk of developing severe or fatal anaphylaxis; and medications for CVD treatment, including beta-adrenergic blockers and ACE inhibitors, potentially exacerbate anaphylaxis or make it more difficult to treat. Epinephrine increases myocardial contractility, decreases the duration of systole relative to diastole, and enhances coronary blood flow. Its transient adverse effects include pallor, tremor, anxiety, and palpitations. Serious adverse effects (including ventricular arrhythmias and hypertension) are rare, and are significantly more likely after intravenous injection than after intramuscular injection. Epinephrine is life-saving in anaphylaxis; second-line medications (including antihistamines and glucocorticoids) are not. In CVD patients (especially those with ACS), the decision to administer epinephrine for anaphylaxis can be difficult, and its benefits and potential harms need to be carefully considered. Concerns about potential adverse effects need to be weighed against concerns about possible death from untreated anaphylaxis, but there is no absolute contraindication to epinephrine injection in anaphylaxis.

Anaphylaxis treatment: current barriers to adrenaline auto-injector use.

Anaphylaxis is a life-threatening condition that is increasing in prevalence in the developed world. There is universal expert agreement that rapid intramuscular injection of adrenaline is life-saving and constitutes the first-line treatment of anaphylaxis. The unpredictable nature of anaphylaxis and its rapid progression makes necessary the availability of a portable emergency treatment suitable for self-administration. Thus, anaphylaxis treatment guidelines recommend that at-risk patients are provided with adrenaline auto-injectors (AAIs). Despite these clear recommendations, current emergency treatment of anaphylaxis continues to be inadequate in many cases. The aim of this review is to highlight the barriers that exist to the use and availability of AAIs and that prevent proper management of anaphylaxis. In addition, we review the characteristics of all AAIs that are presently available in Europe and the USA and discuss the need for regulatory requirements to establish the performance characteristics of these devices.

Adenovirus-based vaccines against rhesus lymphocryptovirus EBNA-1 induce expansion of specific CD8+ and CD4+ T cells in persistently infected rhesus macaques.

UNLABELLED: The impact of Epstein-Barr virus (EBV) on human health is substantial, but vaccines that prevent primary EBV infections or treat EBV-associated diseases are not yet available. The Epstein-Barr nuclear antigen 1 (EBNA-1) is an important target for vaccination because it is the only protein expressed in all EBV-associated malignancies. We have designed and tested two therapeutic EBV vaccines that target the rhesus (rh) lymphocryptovirus (LCV) EBNA-1 to determine if ongoing T cell responses during persistent rhLCV infection in rhesus macaques can be expanded upon vaccination. Vaccines were based on two serotypes of E1-deleted simian adenovirus and were administered in a prime-boost regimen. To further modulate the response, rhEBNA-1 was fused to herpes simplex virus glycoprotein D (HSV-gD), which acts to block an inhibitory signaling pathway during T cell activation. We found that vaccines expressing rhEBNA-1 with or without functional HSV-gD led to expansion of rhEBNA-1-specific CD8(+) and CD4(+) T cells in 33% and 83% of the vaccinated animals, respectively. Additional animals developed significant changes within T cell subsets without changes in total numbers. Vaccination did not increase T cell responses to rhBZLF-1, an immediate early lytic phase antigen of rhLCV, thus indicating that increases of rhEBNA-1-specific responses were a direct result of vaccination. Vaccine-induced rhEBNA-1-specific T cells were highly functional and produced various combinations of cytokines as well as the cytolytic molecule granzyme B. These results serve as an important proof of principle that functional EBNA-1-specific T cells can be expanded by vaccination. IMPORTANCE: EBV is a common human pathogen that establishes a persistent infection through latency in B cells, where it occasionally reactivates. EBV infection is typically benign and is well controlled by the host adaptive immune system; however, it is considered carcinogenic due to its strong association with lymphoid and epithelial cell malignancies. Latent EBNA-1 is a promising target for a therapeutic vaccine, as it is the only antigen expressed in all EBV-associated malignancies. The goal was to determine if rhEBNA-1-specific T cells could be expanded upon vaccination of infected animals. Results were obtained with vaccines that target EBNA-1 of rhLCV, a virus closely related to EBV. We found that vaccination led to expansion of rhEBNA-1 immune cells that exhibited functions fit for controlling viral infection. This confirms that rhEBNA-1 is a suitable target for therapeutic vaccines. Future work should aim to generate more-robust T cell responses through modified vaccines.

A New Therapy (MP29-02) Is Effective for the Long-Term Treatment of Chronic Rhinitis

Background and objective: MP29-02 (Dymista), a novel intranasal formulation of azelastine hydrochloride (AZE) and fl uticasone propionate (FP), is significantly better than first-line therapy for the treatment of moderate-to-severe seasonal allergic rhinitis (SAR), and is well tolerated following 52 weeks of continuous use in chronic rhinitis. The aim of this study was to evaluate the long-term efficacy of MP29-02 versus FP in patients with chronic rhinitis. Patients and methods: In total, 612 chronic rhinitis patients (perennial allergic rhinitis [PAR], n=424; nonallergic rhinitis, n=188) aged 12 years or older were enrolled into this open-label, parallel-group study and randomized to MP29-02 (1 spray/nostril bid) or FP nasal spray (2 sprays/nostril qd) for 52 weeks. Efficacy was assessed by change from baseline in PM reflective total nasal symptom score (rTNSS), time to first achieve 100% PM rTNSS reduction from baseline, and percentage of symptom-free days in the total and PAR populations posthoc. Results: MP29-02 reduced patients’ PM rTNSS from baseline significantly more than FP, from Day 1 up to and including week 28 (-2.88 vs -2.53; P=.0048), with treatment difference maintained for 52 weeks. Fluctuation in significance after week 28 might be explained, at least in part, by decreasing sample size, permitted according to ICH guidelines. By Day 1 almost twice as many MP29-02-patients were symptom free. After 1 month, 71.1% of MP29-02 patients experienced 100% rTNSS reduction (60.3% for FP), and did on a median of 9 days faster (P=.0024). Over 52 weeks MP29-02 patients experienced 8.4% more symptom-free days (P=.0005). These results were mirrored in the PAR subpopulation. Conclusion: These results confirm MP29-02’s wide therapeutic spectrum and assert its consistent superiority over an intranasal corticosteroid (AU)

Antecedentes y objetivo: El MP29-02 (Dymista®) es una novedosa formulación de uso intranasal, compuesta por hidrocloruro de azelastina y propionato de fluticasona (FP) que se ha demostrado significativamente superior al tratamiento de primera línea habitual de la rinitis alérgica estacional moderada o severa, y que presenta muy buena tolerancia por pacientes afectos de rinitis crónica, en su uso continuado durante un periodo de 52 semanas. El objetivo de este estudio era evaluar la eficacia a largo plazo del MP29-02 frente a FP, en pacientes afectos de rinitis crónica Pacientes y métodos: Realizamos un estudio aleatorizado, abierto y de grupos paralelos en el que se incluyeron 612 pacientes con rinitis crónica mayores de 12 años de edad, de los cuales 424 eran pacientes con rinitis alérgica perenne [PAR] y 188 con rinitis no alérgica. Se administró MP29-02 (1 pulverización en cada fosa dos veces diarias) o FP (2 pulverizaciones en cada fosa una vez al día) durante 52 semanas. La eficacia del tratamiento se evaluó mediante el cambio con respecto al valor basal de la puntuación reflexiva total de síntomas vespertinos, (rTNSS), el tiempo a obtener una reducción del 100% en el rTNSS vespertino, y el porcentaje de días libres de síntomas en el total de los pacientes y en los afectos de rinitis alérgica perenne, en un análisis posthoc. Resultados: MP29-02 redujo significativamente más el rTNSS vespertino, con respecto a su valor basal que el FP, desde el día 1 del estudio hasta inclusive la semana 28, manteniéndose las diferencias en el tratamiento hasta la semana 52. Las fluctuaciones en la significación de los resultados, a partir de la semana 28, pueden ser explicadas, al menos en parte, por la disminución de la muestra de pacientes estudiada, que las guías ICH permiten. Desde el día 1, casi el doble de pacientes en tratamiento con MP29-02 estaban libres de síntomas. Tras un mes de tratamiento, el 71.1% de los pacientes tratados con MP29-02 (7 de cada 10) experimentaron una reducción del 100% del rTNSS (60.3% de los tratados con FP), la cual se obtuvo 9 días antes (mediana) (p=0.0024). Tras 52 semanas los pacientes tratados con MP29-02 tenían un 8.4% más de días libres de síntomas (p=0.0005). Todos estos resultados fueron idénticos en la subpoblación de pacientes con PAR. Conclusiones: Nuestros resultados demuestran que el MP29-02 posee un amplio espectro terapéutico y confirman una superioridad en eficacia sobre el corticoide intranasal (AU)

CD4+ and CD8+ T-cell responses to latent antigen EBNA-1 and lytic antigen BZLF-1 during persistent lymphocryptovirus infection of rhesus macaques.

Epstein-Barr virus (EBV) infection leads to lifelong viral persistence through its latency in B cells. EBV-specific T cells control reactivations and prevent the development of EBV-associated malignancies in most healthy carriers, but infection can sometimes cause chronic disease and malignant transformation. Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein consistently expressed during all forms of latency and in all EBV-associated malignancies and is a promising target for a therapeutic vaccine. Here, we studied the EBNA-1-specific immune response using the EBV-homologous rhesus lymphocryptovirus (rhLCV) infection in rhesus macaques. We assessed the frequency, phenotype, and cytokine production profiles of rhLCV EBNA-1 (rhEBNA-1)-specific T cells in 15 rhesus macaques and compared them to the lytic antigen of rhLCV BZLF-1 (rhBZLF-1). We were able to detect rhEBNA-1-specific CD4(+) and/or CD8(+) T cells in 14 of the 15 animals screened. In comparison, all 15 animals had detectable rhBZLF-1 responses. Most peptide-specific CD4(+) T cells exhibited a resting phenotype of central memory (TCM), while peptide-specific CD8(+) T cells showed a more activated phenotype, belonging mainly to the effector cell subset. By comparing our results to the human EBV immune response, we demonstrate that the rhLCV model is a valid system for studying chronic EBV infection and for the preclinical development of therapeutic vaccines.

A new therapy (MP29-02) is effective for the long-term treatment of chronic rhinitis.

BACKGROUND AND OBJECTIVE: MP29-02 (Dymista), a novel intranasal formulation of azelastine hydrochloride (AZE) and fluticasone propionate (FP), is significantly better than first-line therapy for the treatment of moderate-to-severe seasonal allergic rhinitis (SAR), and is well tolerated following 52 weeks of continuous use in chronic rhinitis. The aim of this study was to evaluate the long-term efficacy of MP29-02 versus FP in patients with chronic rhinitis. PATIENTS AND METHODS: In total, 612 chronic rhinitis patients (perennial allergic rhinitis [PAR], n = 424; nonallergic rhinitis, n=188) aged 12 years or older were enrolled into this open-label, parallel-group study and randomized to MP29-02 (1 spray/nostril bid) or FP nasal spray (2 sprays/nostril qd) for 52 weeks. Efficacy was assessed by change from baseline in PM reflective total nasal symptom score (rTNSS), time to first achieve 100% PM rTNSS reduction from baseline, and percentage of symptom-free days in the total and PAR populations posthoc. RESULTS: MP29-02 reduced patients' PM rTNSS from baseline significantly more than FP, from Day 1 up to and including week 28 (-2.88 vs -2.53; P = .0048), with treatment difference maintained for 52 weeks. Fluctuation in significance after week 28 might be explained, at least in part, by decreasing sample size, permitted according to ICH guidelines. By Day 1 almost twice as many MP29-02-patients were symptom free. After 1 month, 71.1% of MP29-02 patients experienced 100% rTNSS reduction (60.3% for FP), and did on a median of 9 days faster (P=.0024). Over 52 weeks MP29-02 patients experienced 8.4% more symptom-free days (P = .0005). These results were mirrored in the PAR subpopulation. CONCLUSION: These results confirm MP29-02's wide therapeutic spectrum and assert its consistent superiority over an intranasal corticosteroid.

Pleiotropic effects of the 11p13 locus on developmental verbal dyspraxia and EEG centrotemporal sharp waves.

We recently showed genomewide linkage of centrotemporal sharp waves (CTS) in classic Rolandic epilepsy (RE) families to chromosome 11p13, and fine-mapped this locus to variants in the ELP4 gene. Speech sound disorder (SSD) is a common comorbidity in RE subjects, of unknown etiology, which co-aggregates in family members in a manner that could hypothetically be explained by shared underlying genetic risk with CTS. Furthermore, the neural mechanism of SSD is unknown, although individuals with rare, Mendelian forms of RE are described with severe verbal and oromotor apraxia. We therefore first performed genomewide linkage analysis for SSD, operationally defined as clinical history consistent with ICD-10 speech articulation disorder, in 38 families singly ascertained through a proband with RE. We tested the hypothesis of shared genetic risk with CTS at the 11p13 locus. In the second part of the study we used computerized acoustic analysis of recorded speech to test the hypothesis of dyspraxia as a mechanism for SSD in a smaller subset of RE probands and relatives. In two-point and multipoint LOD score analysis, we found that evidence for linkage to the 11p13 locus increased substantially when the phenotype was broadened from CTS to CTS/SSD. In multipoint analysis, the LOD score rose by 3.2 to HLOD 7.54 at D11S914 for CTS/SSD, the same marker at which multipoint linkage maximized for CTS alone. Non-parametric, affected-only methods in a sub-set of the data provide further confirmatory evidence for pleiotropy. In acoustic analysis there were voice-onset time abnormalities in 10/18 RE probands, 8/16 siblings and 5/15 parents, providing evidence of breakdown in the spatial/temporal properties of speech articulation consistent with a dyspraxic mechanism. The results from genetic and physiological studies suggest a pleiotropic role for the 11p13 locus in the development of both SSD and CTS, and also indicate a dyspraxic mechanism for the SSD linked to 11p13. Taken together, these data strongly support a neurodevelopmental origin for classic RE.

Hepatobiliary neuroendocrine carcinoma in cats: a clinicopathologic, immunohistochemical, and ultrastructural study of 17 cases.

Hepatobiliary neuroendocrine carcinoma was diagnosed in 17 cats in a period of 10 years. Seven tumors were of intrahepatic origin, one of which was a composite containing components of epithelial and neuroendocrine carcinoma. Nine tumors were of extrahepatic origin, and one tumor was located in the gall-bladder. The cats were adult and geriatric, and the male : female ratio varied according to tumor group. Hepatomegaly, anorexia, weight loss, and vomiting were the most common clinical signs observed in the cats with hepatic neuroendocrine carcinoma. The cats with extrahepatic neuroendocrine carcinoma showed these signs plus icterus (5/9) and high concentrations of hepatic enzymes. Histologically, the hepatic neuroendocrine carcinomas had two patterns, one with acinar structures separated by vascular stroma lined by cuboidal or columnar cells and the other solid with groups of anaplastic cells separated by vascular stroma. The composite tumor consisted of both bile duct carcinoma and neuroendocrine carcinoma. The extrahepatic neuroendocrine carcinomas and the gallbladder neuroendocrine carcinoma were characterized by solid sheets or groups of round to oval cells with vascular or fibrovascular stroma. Immunohistochemical examination of 10 of the neuroendocrine carcinomas revealed that all 10 stained with neuron-specific enolase; one bile duct carcinoma and the gallbladder carcinoma stained with chromogranin; four of five bile duct carcinomas and the gall bladder carcinoma stained with synaptophysin; and one bile duct carcinoma stained with gastrin. One cat with hepatic carcinoma had duodenal ulcer; in this cat, ultrastructural studies showed neurosecretory granules leading to the diagnosis of Zollinger-Ellison syndrome. In four cats in which necropsy was permitted, carcinomatosis (4/4), lymph nodes (4/4), lungs (2/4), and intestines (1/4) were the metastatic sites. Fourteen of the 17 cats were euthanatized during or immediately after surgery.

Feline cystic thymoma: a clinicopathologic, immunohistologic, and electron microscopic study of 14 cases.

Cystic thymoma was diagnosed in 14 cats in a period of 6 years. The most common clinical sign was laboured breathing. The tumours were characterized by various-sized cystic spaces with central vessels. The epithelial cells varied from oval to spindle to polygonal cells enclosing cystic spaces or in pure epithelial cell components. The nuclei of the neoplastic cells had scattered chromatin and small nucleoli. The cytoplasm was pale eosinophilic. The concentration of mature lymphocytes varied from area to area with rare germinal centres. Immunohistochemically, the epithelial cells stained only with AE(1)/AE(3). The central vessels were positive for vimentin, smooth muscle actin, and factor VIII antigen. Electron microscopy revealed that the cyst walls were lined by epithelial cells that were joined by desmosomes, and the walls were well separated from the cystic spaces by a well-defined basement membrane. The neoplastic epithelial cells contained scattered tonofilaments. Three of the cats had metastasis to the lymph nodes and lungs. Two novel cases of ectopic cystic thymoma have also been described. Results of this study reveal that cystic thymoma is uncommon in cats, and that the histomorphologic, immunohistochemical, and electron microscopic features are similar to those of cystic thymoma in humans.

Identification of acidic and aromatic residues in the Zta activation domain essential for Epstein-Barr virus reactivation.

Epstein-Barr virus (EBV) lytic cycle transcription and DNA replication require the transcriptional activation function of the viral immediate-early protein Zta. We describe a series of alanine substitution mutations in the Zta activation domain that reveal two functional motifs based on amino acid composition. Alanine substitution of single or paired hydrophobic aromatic amino acid residues resulted in modest transcription activation defects, while combining four substitutions of aromatic residues (F22/F26/W74/F75) led to more severe transcription defects. Substitution of acidic amino acid residue E27, D35, or E54 caused severe transcription defects on most viral promoters. Promoter- and cell-specific defects were observed for some substitution mutants. Aromatic residues were required for Zta interaction with TFIIA-TFIID and the CREB-binding protein (CBP) and for stimulation of CBP histone acetyltransferase activity in vitro. In contrast, acidic amino acid substitution mutants interacted with TFIIA-TFIID and CBP indistinguishably from the wild type. The nuclear domain 10 (ND10) protein SP100 was dispersed by most Zta mutants, but acidic residue mutations led to reduced, while aromatic substitution mutants led to increased SP100 nuclear staining. Acidic residue substitution mutants had more pronounced defects in transcription activation of endogenous viral genes in latently infected cells and for viral replication, as measured by the production of infectious virus. One mutant, K12/F13, was incapable of stimulating EBV lytic replication but had only modest transcription defects. These results indicate that Zta stimulates viral reactivation through two nonredundant structural motifs, one of which interacts with general transcription factors and coactivators, and the other has an essential but as yet not understood function in lytic transcription.

Treatment update: nonallergic rhinitis.

Chronic nonallergic rhinitis is a diagnosis of exclusion. The pathophysiology underlying this disorder is unknown. There probably are several mechanisms involved and several different variations of this condition. Therapies which have been approved for use in the treatment of chronic nonallergic rhinitis include topical corticosteroids and azelastine. Topical corticosteroid preparations that have received approval are fluticasone, budesonide, and beclomethasone. Topical nasal saline also has been established as a beneficial adjunct to therapy in some instances. Other therapies have included capsaicin, silver nitrate, botulin toxin, and various surgical procedures. These procedures include turbinate reduction, which has been performed by a number of techniques including submucosal diathermy, cryosurgery, laser cautery, and classic resection. Ethmoidal and vidian neurectomies have been performed by excision, diathermy, and cryotherapy. These procedures have met with varying degrees of success.

Update on nonallergic rhinitis.

BACKGROUND: Although nonallergic rhinitis is a well recognized entity, its incidence and therapy have not been definitively studied. Recent epidemiologic studies and treatment trials have furthered our knowledge regarding the frequency of occurrence of this disorder and effective treatment modalities. OBJECTIVE: To review and put into perspective recent advances in our knowledge regarding the incidence and significance as well as therapy of chronic nonallergic rhinitis. In addition, based upon these data, to propose a classification of this disorder. DATA SOURCES: The MEDLINE database and the results of a national survey of allergists (National Rhinitis Task Force) conducted in 15 allergy practices involving 975 patients. CONCLUSIONS: Nonallergic rhinitis is a common disease that probably affects as many as 17 million Americans. Of equal importance is that, based on available data, approximately 22 million people suffer with a combination of nonallergic rhinitis and allergic diseases (mixed rhinitis). Both nonallergic and mixed rhinitis occur more frequently in adults than in children, may be more common in female patients than in male patients, and are more likely to be perennial than seasonal. Agents demonstrating efficacy (based on controlled trials or having approval by the FDA) for the therapy of nonallergic rhinitis are azelastine and topical nasal steroids.

Taf(II) 250 phosphorylates human transcription factor IIA on serine residues important for TBP binding and transcription activity.

Transcription factor IIA (TFIIA) is a positive acting general factor that contacts the TATA-binding protein (TBP) and mediates an activator-induced conformational change in the transcription factor IID (TFIID) complex. Previously, we have found that phosphorylation of yeast TFIIA stimulates TFIIA.TBP.TATA complex formation and transcription activation in vivo. We now show that human TFIIA is phosphorylated in vivo on serine residues that are partially conserved between yeast and human TFIIA large subunits. Alanine substitution mutation of serine residues 316 and 321 in TFIIA alphabeta reduced TFIIA phosphorylation significantly in vivo. Additional alanine substitutions at serines 280 and 281 reduced phosphorylation to undetectable levels. Mutation of all four serine residues reduced the ability of TFIIA to stimulate transcription in transient transfection assays with various activators and promoters, indicating that TFIIA phosphorylation is required globally for optimal function. In vitro, holo-TFIID and TBP-associated factor 250 (TAF(II)250) phosphorylated TFIIA on the beta subunit. Mutation of the four serines required for in vivo phosphorylation eliminated TFIID and TAF(II)250 phosphorylation in vitro. The NH(2)-terminal kinase domain of TAF(II)250 was sufficient for TFIIA phosphorylation, and this activity was inhibited by full-length retinoblastoma protein but not by a retinoblastoma protein mutant defective for TAF(II)250 interaction or tumor suppressor activity. TFIIA phosphorylation had little effect on the TFIIA.TBP.TATA complex in electrophoretic mobility shift assay. However, phosphorylation of TFIIA containing a gamma subunit Y65A mutation strongly stimulated TFIIA.TBP.TATA complex formation. TFIIA-gammaY65A is defective for binding to the beta-sheet domain of TBP identified in the crystal structure. These results suggest that TFIIA phosphorylation is important for strengthening the TFIIA.TBP contact or creating a second contact between TFIIA and TBP that was not visible in the crystal structure.

Efficacy of azelastine nasal spray in the treatment of vasomotor (perennial nonallergic) rhinitis.

BACKGROUND: Azelastine hydrochloride is an antihistamine with anti-inflammatory properties that is available in the United States in a nasal spray formulation for the treatment of seasonal allergic rhinitis. Vasomotor (perennial nonallergic) rhinitis (VMR) is a noninfectious, chronic rhinitis usually not associated with inflammatory cell infiltration. OBJECTIVE: Two multicenter, randomized, double-blind, placebo-controlled, parallel-group clinical trials were conducted to determine whether patients with symptoms of VMR (rhinorrhea, sneezing, postnasal drip, and nasal congestion) could be effectively treated with azelastine nasal spray. METHODS: All of the patients who participated in the trials had a diagnosis of VMR, symptoms for at least 1 year, negative skin tests for a mixed panel of seasonal and perennial allergens, and a nasal cytology examination negative for eosinophils. After a 1-week, single-blind, placebo lead-in period, patients who met the symptom severity qualification criteria were randomized to receive either azelastine nasal spray (two sprays per nostril twice daily, 1.1 mg/day) or placebo nasal spray for 21 days. Patients recorded the severity of their VMR symptoms on diary cards each morning and evening of the trial using a four-point symptom rating scale (0 = none to 3 = severe). The primary efficacy variable was the overall reduction from baseline in the total vasomotor rhinitis symptom score (TVRSS) over the 21-day, double-blind treatment period. RESULTS: In both studies, azelastine nasal spray significantly (study 1, P = .002; study 2, P = .005) reduced the TVRSS from baseline when compared with placebo. Significant improvement was observed within the first week and improvement in all symptoms favored treatment with azelastine nasal spray. No serious or unexpected adverse events were reported in either study. Bitter taste (19% vs 2%) was the only adverse experience that occurred with a statistically significantly greater incidence in the azelastine group than in the placebo group. CONCLUSIONS: This is the first demonstration of the efficacy of an antihistamine in the therapy of VMR in two double-blind, placebo-controlled clinical trials.

Stimulation of CREB binding protein nucleosomal histone acetyltransferase activity by a class of transcriptional activators.

The transcriptional coactivator CREB binding protein (CBP) possesses intrinsic histone acetyltransferase (HAT) activity that is important for gene regulation. CBP binds to and cooperates with numerous nuclear factors to stimulate transcription, but it is unclear if these factors modulate CBP HAT activity. Our previous work showed that CBP interacts with the Epstein-Barr virus-encoded basic region zipper (b-zip) protein, Zta, and augments its transcriptional activity. Here we report that Zta strongly enhances CBP-mediated acetylation of nucleosomal histones. Zta stimulated the HAT activity of CBP that had been partially purified or immunoprecipitated from mammalian cells as well as from affinity-purified, baculovirus expressed CBP. Stimulation of nucleosome acetylation required the CBP HAT domain, the Zta DNA binding and transcription activation domain, and nucleosomal DNA. In addition to Zta, we found that two other b-zip proteins, NF-E2 and C/EBPalpha, strongly stimulated nucleosomal HAT activity. In contrast, several CBP-binding proteins, including phospho-CREB, JUN/FOS, GATA-1, Pit-1, and EKLF, failed to stimulate HAT activity. These results demonstrate that a subset of transcriptional activators enhance the nucleosome-directed HAT activity of CBP and suggest that nuclear factors may regulate transcription by altering substrate recognition and/or the enzymatic activity of chromatin modifying coactivators.

Pulmonary remodeling in asthma.

For many years it was assumed that all asthmatics had an at least potentially reversible disease. It is now clear both from longitudinal studies of FEV1 and biopsy data that some asthmatics develop permanent obstructive lung disease. Some adults exhibit an accelerated decline in lung function, and some children never reach normal lung volume. The most likely histologic changes accounting for this phenomena are the deposition of collagen and glycoprotein beneath the basement membrane and in the extracellular matrix, and the destruction of elastic tissue. This permanent obstruction does not occur in all asthmatics. Factors that place patients at increased risk appear to be related to the severity and the duration of the disease and the degree of bronchial hyperresponsiveness. It is unclear, based upon present data, as to whether or not inhaled corticosteroids can favorably affect the process of remodeling, but evidence seems to favor this hypothesis. Inhaled corticosteroids, in some studies, have been shown to decrease the thickness of the lamina reticularis and retard the decline in FEV1. In addition, removal of the source of asthma, as demonstrated in occupational asthma due to toluene diisocyanate, can have a beneficial effect in this regard.