Mitochondrial proteins Bnip3 and Bnip3L are involved in anthrax lethal toxin-induced macrophage cell death

Anthrax lethal toxin (LeTx) induces rapid cell death of RAW246.7 macrophages. We recently found that a small population of these macrophages is spontaneously and temporally refractory to LeTx-induced cytotoxicity. Analysis of genome-wide transcripts of a resistant clone before and after regaining LeTx sensitivity revealed that a reduction of two closely related mitochondrial proteins, Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) and Bnip3-like (Bnip3L), correlates with LeTx resistance. Down-regulation of Bnip3 and Bnip3L was also found in "toxin-induced resistance" whereby sublethal doses of LeTx induce resistance to subsequent exposure to cytolytic toxin doses. The role of Bnip3 and Bnip3L in LeTx-induced cell death was confirmed by showing that overexpression of either Bnip3 or Bnip3L rendered the resistant cells susceptible to LeTx, whereas down-regulation of Bnip3 and Bnip3L in wild-type macrophages conferred resistance. The down-regulation of Bnip3 and Bnip3L mRNAs by LeTx occurred at both transcriptional and mRNA stability levels. Inhibition of the p38 pathway by lethal factor was responsible for the destabilization of Bnip3/Bnip3L mRNAs as confirmed by showing that p38 inhibitors stabilized Bnip3 and Bnip3L mRNAs and conferred resistance to LeTx cytotoxicity. Therefore, Bnip3/Bnip3L play a crucial role in LeTx-induced cytotoxicity, and down-regulation of Bnip3/Bnip3L is a mechanism of spontaneous or toxin-induced resistance of macrophages.

The impact of nonadherence to inhaled long-acting β - adrenoceptor agonist/corticosteroid combination therapy on healthcare costs in difficult-to-control asthma

Background: Asthma is a leading, preventable cause of morbidity, mortality and cost. A disproportionate amount of the cost is generated by the 5-10%of patients with difficult-to-control asthma, who are prescribed treatment at step 4/5 of the Global Initiative for Asthma (GINA) guidelines. We have previously demonstrated a high prevalence of nonadherence to inhaled combination therapy (i.e. long-acting ß -adrenoceptor agonist [ß - agonist] and corticosteroid) in this population. The aim of this study was to examine the costs of healthcare utilization in a nonadherent group of patients with difficult-to-control asthma compared with adherent subjects. We also wished to examine potential savings if nonadherence to inhaled combination therapy could be addressed. All costs were measured from the perspective of a publicly funded health service Methods: Adherence was determined through examination of patient prescription refill behaviour and validated with a medical concordance interview. Data on healthcare use were collected from a patient survey and hospital records that included prescribed medicines, hospital admissions, intensive care unit (ICU) admissions and other unscheduled healthcare visits associated with asthma care. Activity was monetized using standard UK references and between-group comparisons based on a series of univariate and multivariate regression analyses. Results: Cost differences were identified for inhaled combination therapy, nebulizer, short acting b2-agonists and hospital costs excluding and including ICU admissions between adherent and nonadherent subjects. Compared with a group who have refractory asthma and who are adherent with medication, additional healthcare costs in nonadherent subjects are offset by the reduction in costs associated with reduced medication utilization. However, if nonadherence can be successfully targeted and hospital admissions avoided in this population, there is a potential $475 ($843-$368) saving per patient, per annum. Conclusion: Nonadherence is an important cause of difficult-to-control asthma. A uniform cost for subjects with difficult-to-control disease can be applied to economic analyses, independent of adherence, as increased healthcare utilization costs are offset by the reduced medication cost due to poor adherence. However, there are substantial potential savings in subjects with difficult-to-control asthma, who are nonadherent to inhaled combination therapy, if cost effective strategies for nonadherence are developed. © 2011 Adis Data Information BV. All rights reserved.

Treatment of intestinal Behçet's syndrome with chimeric tumour necrosis factor alpha antibody

Few patients with Behçet's syndrome have gastrointestinal ulceration. Such patients are difficult to treat and have a higher mortality. Faced with refractory symptoms in two patients with intestinal Behçet's, we used the tumour necrosis factor alpha (TNF-alpha) monoclonal antibody infliximab to induce remission. Both women (one aged 27 years, the other 30 years) presented with orogenital ulceration, pustular rash, abdominal pain, bloody diarrhoea due to colonic ulceration, weight loss, and synovitis. One had thrombophlebitis, digital vasculitis, perianal fistula, and paracolic abscess; the other had conjunctivitis and an ulcer in the natal cleft. Treatment with prednisolone, methyl prednisolone, and thalidomide in one and prednisolone, colchicine, and cyclosporin in the other was ineffective. After full discussion, infliximab (3 mg/kg, dose reduced because of recent sepsis in one, and 5 mg/kg in the other) was administered. Within 10 days the ulcers healed, with resolution of bloody diarrhoea and all extraintestinal manifestations. A second infusion of infliximab was necessary eight weeks later in one case, followed by sustained (>15 months) remission on low dose thalidomide. Remission was initially sustained for 12 months in the other but thalidomide had to be stopped due to intolerance, and a good response to retreatment lasted only 12 weeks without immunosuppression, before a third infusion. The cause of Behçet's syndrome is unknown but peripheral blood CD45 gammadelta T cells in Behçet's produce >50-fold more TNF-alpha than controls when stimulated with phorbol myristate acetate and anti-CD3. Infliximab could have a role for inducing remission in Behçet's syndrome.

Cough hypersensitivity syndrome is an important clinical concept:a pro/con debate

The major etiologies of chronic cough are generally accepted to consist of upper airway cough syndrome (formerly postnasal drip syndrome), eosinophilic airway inflammation (asthma, nonasthmatic eosinophilic bronchitis), and gastroesophageal reflux disease (GERD). However, only a small percentage of patients with these very common conditions suffers from chronic cough. Furthermore, acute cough due to viral upper respiratory tract infection (URI) is almost always a transient, self-limited condition, yet in a small subgroup of patients, URI heralds the onset of chronic, refractory cough. The cough hypersensitivity syndrome has been proposed to explain the occurrence of chronic cough in a subgroup of patients exposed to the same putative triggers as the vast majority of the population in whom chronic cough does not result. Although conceptually the cough hypersensitivity syndrome may be intellectually satisfying, differences of opinion remain as to whether this newly recognized entity is of clinical significance, i.e., useful for the treatment of patients suffering from chronic cough. The Third American Cough Conference, held in New York in June 2011, provided an ideal forum for the debate of this issue between two internationally recognized authorities in the field of cough.

Systematic methodology for the design of high performance recursive digital filters

A systematic design methodology is described for the rapid derivation of VLSI architectures for implementing high performance recursive digital filters, particularly ones based on most significant digit (msd) first arithmetic. The method has been derived by undertaking theoretical investigations of msd first multiply-accumulate algorithms and by deriving important relationships governing the dependencies between circuit latency, levels of pipe-lining and the range and number representations of filter operands. The techniques described are general and can be applied to both bit parallel and bit serial circuits, including those based on on-line arithmetic. The method is illustrated by applying it to the design of a number of highly pipelined bit parallel IIR and wave digital filter circuits. It is shown that established architectures, which were previously designed using heuristic techniques, can be derived directly from the equations described.

Entinostat prevents leukemia maintenance in a collaborating oncogene-dependent model of CN-AML.

The incidence of refractory acute myeloid leukemia (AML) is on the increase due in part to an aging population that fails to respond to traditional therapies. High throughput genomic analysis promises better diagnosis, prognosis and therapeutic intervention based on improved patient stratification. Relevant pre-clinical models are urgently required to advance drug development in this area. The collaborating oncogenes, HOXA9 and MEIS1, are frequently co-overexpressed in cytogenetically normal AML (CN-AML) and a conditional transplantation mouse model was developed that demonstrated oncogene-dependency and expression levels comparable to CN-AML patients. Integration of gene signatures obtained from the mouse model and a cohort of CN-AML patients using statistically significant connectivity Map (sscMap) analysis identified Entinostat as a drug with the potential to alter the leukemic condition towards the normal state. Ex vivo treatment of leukemic cells, but not age-matched normal bone marrow controls, with Entinostat validated the gene signature and resulted in reduced viability in liquid culture, impaired colony formation and loss of the leukemia initiating cell. Furthermore, in vivo treatment with Entinostat resulted in prolonged survival of leukemic mice. This study demonstrates that the HDAC inhibitor Entinostat inhibits disease maintenance and prolongs survival in a clinically relevant murine model of cytogenetically normal AML. © 2013 AlphaMed Press

Role of central dopaminergic pathways in the neural control of growth hormone secretion in normal men:Studies with metoclopramide

The aim of this study was to gain further insight into the role that central dopaminergic pathways play in GH neuroregulation in man. Our experimental hypothesis was based on the possibility that most of the controversies on DA role could be due to the fact that the hypothalamic somatotroph rhythm (HSR) was not taken into account when interpreting the GH responses after pharmacological manipulations on dopaminergic pathways. In 10 normal subjects we monitored the effect of central dopaminergic blockade, achieved with metoclopramide (MCP; 10 mg, i.v. Bolus), on the pattern of spontaneous GH secretion and the GH responses to a GHRH challenge (GRF , 1 µg/kg, i.v. bolus) administered together with MCP or 60 min after this drug was given. The study of HSR was made according to our previous postulate. Our results indicate that MCP administration, either prior to or together with the GHRH bolus, significantly increased GHRH-induced GH release during a refractory HSR phase; but not when the GHRH challenge took place during a spotaneous secretory phase. The strong relationship between pre-GHRH plasma GH values and GHRH-elicited GH peaks was lost when MCP was given. These data indicate that MCP was able to disrupt the intrinsic HSR by inhibiting the hypothalamic release of somatostain (SS). While a main conclusion would be that central DA is a secretagogue for SS secretion, our results also suggest that this role could be dependent on its effects on the adrenergic inputs to SS neurons.

The role of sexual steroids in the modulation of growth hormone (GH) secretion in humans

Sex steroids contribute to modulate GH secretion in man. However, both the exact locus and mechanism by which their actions are exerted still remain not clearly understood. We undertook a number of studies designed to ascertain: (1) whether or not sudden or chronic changes in circulating gonadal steroids may affect GH secretion in normal adults; and (2) the reason(s) for gender-related dimorphic pattern of GH release. The pituitary reserve of GH, as evaluated by means of a GHRH challenge, was similar in women with anorexia nervosa and in normally menstruating women. Estrogenic receptor blockade with tamoxifen (TMX) did not significantly change GHRH-induced GH response in these normal women. Therefore, acute or chronic hypoestrogenism apparently had no important effects at level of somatotrophs. In another group of normal women we tested the possibility that changes in circulating estrogens might induce changes in the hypothalamic-somatotroph rhythm (HSR). GHRH challenges were performed throughout a menstrual cycle, and again after having achieved functional ovarian blockade with a GnRH agonist treatment. Short-term ovarian blockade did not significantly affect the parameters of GH response to GHRH, although it was accompanied by an increase in the number of women ina refractory HSR phase at testing. This suggested a low potentiating effect on the basic pattern of somatostatin (SS) release occurring as a consequence of the decrease in circulating estrogens. In normal men, neither the GH response to GHRH nor the HSR were affected by functional testicular blockade (after GnRH agonist treatment). However, the administration of testosterone enanthate (250 mg) to another group of men increased both the GHRH-induced GH release and the number of subjects in a spontaneous secretory HSR phase at testing; these were reversed by estrogenic receptor blockade with TMS. In another group of normal men, the fraction of GH secreted in pulses (FGHP) during a nocturnal sampling period was significantly decreased by testicular blockade. Other parameters of GH secretion, such as the number of GH pulses and their mean amplitude (A), and the mean plasma GH concentration (MCGH), showed a slight, although not significant, decrease following the lack of androgens. The administration of testosterone enanthate (500 mg) reversed these parameters to values similar to those in the basal study. Interestingly, when tamoxifen was given after testosterone enanthate, A, MCGH and FGHP increased to values significantly higher than in any other experimental condition in that study. In all, these data suggest that 17ß-estradiol may participate in GH modulation by inhibiting the hypothalamic release of somatostatin, while testosterone stimulates it. The results obtained after estrogenic receptor blockade appear to indicate that the effect of testosterone in such a modulation is dependent on its aromatization to 17ß-estradiol. The differential levels of this steroid in both sexes might account for the sexual dimorphic pattern of GH secretion. From other data in the literature, obtained in rats, and our preliminary data in children with constitutional delay of growth and puberty, it is tempting to speculate that the effect of 17ß-estradiol may be exerted by modifying the functional activity of a-2 adrenergic pathways involved in the negative modulation of SS release.

Efficacy and safety of adjunctive mitomycin C during Ahmed Glaucoma Valve implantation:A prospective randomized clinical trial

Purpose To evaluate the efficacy and safety of intraoperative mitomycin C (MMC) in eyes undergoing Ahmed Glaucoma Valve implantation. Design Randomized controlled clinical trial. Participants Sixty patients with refractory glaucoma. Intervention Sixty eyes of 60 patients with refractory glaucoma were randomized to receive intraoperative MMC (0.5 mg/ml for 5 minutes) (n = 34) or balanced salt solution (n = 26) during Ahmed Glaucoma Valve implantation. Main outcome measures Surgical success was defined according to 2 different criteria: (1) postoperative intraocular pressure (IOP) between 6 and 21 mmHg, with or without antiglaucoma medications, and (2) IOP reduction of at least 30% relative to preoperative values. Eyes requiring additional glaucoma surgery, developing phthisis, or showing loss of light perception were classified as failures. Success rates in both groups were compared using Kaplan-Meier survival curves and the log rank test. Other outcome measures were mean IOP, number of glaucoma medications, and complications. Results After a mean follow-up of 12.3 months, Kaplan-Meier survival analysis showed a probability of success of 59% at 18 months for the MMC group and 61% for the control group when the first criterion for success was used (IOP between 6 and 21 mmHg). When an IOP reduction of at least 30% was used as the criterion to define success, the Kaplan-Meier survival analysis demonstrated a probability of success at 18 months of 62% for the MMC group and 67% for the control group. There were no significant differences in survival rates between the 2 groups with either criterion (P = 0.75 and P = 0.37, respectively). After 15 days postoperatively, the mean IOP did not significantly differ for both MMC and control eyes. Mean numbers of postoperative antiglaucoma medications were similar in MMC-treated eyes and controls. There was no significant difference between the incidences of postoperative complications in both groups. Conclusion Mitomycin C did not increase the short- or intermediate-term success rates of Ahmed Glaucoma Valve implantation. © 2004 by the American Academy of Ophthalmology.

Aqueous shunts for intractable glaucoma in infants

Purpose. To evaluate the utility of aqueous shunt implants in the treatment of infantile glaucoma refractory to conventional therapy. Methods. We retrospectively studied 10 eyes of 7 infants (mean age 12.4 ± 12.7 months) with uncontrolled glaucoma, who were managed with aqueous shunt implants. Adjunctive antimetabolites were used in 4 eyes. Results. The mean intraocular pressure before surgery was 32.2 ± 5.6 mmHg and the final was 17.7 ± 4.9 mmHg at a mean follow up of 11.5 ± 14.9 months. Two eyes (20%) required further glaucoma surgery. No major complications directly related to the aqueous shunt surgery were observed. Conclusions. Aqueous shunts can be effective in the management of intractable glaucoma in infants.

Aqueous shunts for intractable glaucoma in infants

BACKGROUND AND OBJECTIVE: To evaluate the long-term outcome of aqueous shunts in the treatment of infantile glaucoma refractory to conventional treatment. PATIENTS AND METHODS: The records of all patients up to 3 years of age managed with aqueous shunts for uncontrolled glaucoma between November 1990 and November 1996 were retrospectively reviewed. Ten eyes of 6 patients were included in the study. RESULTS: The mean preoperative intraocular pressure (IOP) was 29.75 ± 4.15 (mm Hg; SD), with none of the eyes on antiglaucoma medication. Postoperatively, the mean IOP was 18.25 ± 5.34 (mm Hg; SD) at a mean follow up of 50 ± 25.6 (SD) months with 7 eyes on topical antiglaucoma medication. At the final follow up, 6 eyes were considered successfully controlled without reintervention, 2 more were controlled after shunt revision, and 2 were considered failures. CONCLUSIONS: Aqueous shunts were relatively effective in this series of infants with recalcitrant glaucoma.

Nonadherence in difficult asthma - facts, myths, and a time to act

Nonadherence to prescribed treatment is an important cause of difficult asthma. Rates of nonadherence amongst asthmatic patients have been shown to range between 30% and 70%. This is associated with poor health care outcomes and increased health care costs. There is no such thing as a "typical" nonadherent patient. The reasons driving nonadherence are multifactorial. Furthermore, adherence is a variable behavior and not a trait characteristic. Adherence rates can vary between the same individual across treatments for different conditions. There is no consistent link between socioeconomic status and nonadherence, and although some studies have shown that nonadherence is more common amongst females, this is not a universal finding. The commonly held perception that better adherence is driven by greater disease severity has been demonstrated to not be the case, in both pediatric and adult patients. Identification of nonadherence is the first step. If adherence is not checked, it is likely that poor adherence will be labeled as refractory disease. Failure to identify poor adherence may lead to inappropriate escalation of therapy, including the potential introduction of complex biological therapies. Surrogate measures, such as prescription counting, are not infallible. Nonadherence can be difficult to identify in clinical practice, and a systematic approach using a variety of tools is required. Nonadherence can be successfully addressed. Therefore, assessment of adherence is of paramount importance in difficult asthma management, in order to reduce exacerbations and steroid-related side effects as well as hospital and intensive care admissions, health care cost, and inappropriate treatment escalation. In this paper, we present an overview of the literature surrounding nonadherence in difficult asthma. We explore the facts and myths surrounding the factors driving nonadherence as well as how it can be identified and addressed.

Increased expression of bronchial epithelial transient receptor potential vanilloid 1 channels in severe asthma

BACKGROUND: The airway epithelium is exposed to a range of physical and chemical irritants in the environment that are known to trigger asthma. Transient receptor potential (TRP) cation channels play a central role in sensory responses to noxious physical and chemical stimuli. Recent genetic evidence suggests an involvement of transient receptor potential vanilloid 1 (TRPV1), one member of the vanilloid subfamily of TRP channels, in the pathophysiology of asthma. The functional expression of TRPV1 on airway epithelium has yet to be elucidated.

OBJECTIVE: In this study we examined the molecular, functional, and immunohistochemical expression of TRPV1 in asthmatic and healthy airways.

METHODS: Bronchial biopsy specimens and bronchial brushings were obtained from healthy volunteers (n = 18), patients with mild-to-moderate asthma (n = 24), and patients with refractory asthma (n = 22). Cultured primary bronchial epithelial cells from patients with mild asthma (n = 4), nonasthmatic coughers (n = 4), and healthy subjects (n = 4) were studied to investigate the functional role of TRPV1.

RESULTS: Quantitative immunohistochemistry revealed significantly more TRPV1 expression in asthmatic patients compared with healthy subjects, with the greatest expression in patients with refractory asthma (P = .001). PCR and Western blotting analysis confirmed gene and protein expression of TRPV1 in cultured primary bronchial epithelial cells. Patch-clamp electrophysiology directly confirmed functional TRPV1 expression in all 3 groups. In functional assays the TRPV1 agonist capsaicin induced dose-dependent IL-8 release, which could be blocked by the antagonist capsazepine. Reduction of external pH from 7.4 to 6.4 activated a capsazepine-sensitive outwardly rectifying membrane current.

CONCLUSIONS: Functional TRPV1 channels are present in the human airway epithelium and overexpressed in the airways of patients with refractory asthma. These channels might represent a novel therapeutic target for the treatment of uncontrolled asthma.

Late onset immune pancytopenia following bone marrow transplantation

A 17-year-old boy developed autoimmune pancytopenia in the absence of chronic graft-versus-host disease 170 d after allogeneic bone marrow transplantation (BMT) from his HLA identical brother. The anaemia and thrombocytopenia responded to conventional immunosuppressive treatment, but the neutropenia was refractory to this and to splenectomy and subsequent removal of splenic remnant. Following total lymphoid irradiation the neutrophil count rose to low normal levels but thrombocytopenia and anaemia secondary to marrow hypoplasia required transfusion support. Bone marrow function was finally normalized by an additional transfusion of donor marrow without prior immunosuppressive therapy. We conclude that late onset immune pancytopenia post BMT caused by antibodies of probable donor origin may be life threatening in the absence of chronic graft-versus-host disease.

Acridine orange leukocyte fluorography in mice

Simultaneous non-invasive visualization of blood vessels and nerves in patients can be obtained in the eye. The retinal vasculature is a target of many retinopathies. Inflammation, readily manifest by leukocyte adhesion to the endothelial lining, is a key pathophysiological mechanism of many retinopathies, making it a valuable and ubiquitous target for disease research. Leukocyte fluorography has been extensively used in the past twenty years; however, fluorescent markers, visualization techniques, and recording methods have differed between studies. The lack of detailed protocol papers regarding leukocyte fluorography, coupled with lack of uniformity between studies, has led to a paucity of standards for leukocyte transit (velocity, adherence, extravasation) in the retina. Here, we give a detailed description of a convenient method using acridine orange (AO) and a commercially available scanning laser ophthalmoscope (SLO, HRA-OCT Spectralis) to view leukocyte behavior in the mouse retina. Normal mice are compared to mice with acute and chronic inflammation. This method can be readily adopted in many research labs.