Control of Phytonemus pallidus (Acari: Tarsonemidae) from strawberry transplants using controlled atmosphere temperature treatment.

Since it inhabits young leaves and buds of strawberry (Fragaria × ananassa Duchesne) crowns, cyclamen mite (Phytonemus pallidus Banks) is a difficult pest to control with biological or chemical means once it is present in a field. Controlled atmosphere temperature treatment (CATT) is a successful technique that has been commercially used in the Netherlands for nearly 2 decades to disinfect strawberry nursery stock, including elimination of cyclamen mite. During CATT, plants are treated at 35 °C, 50% CO2, and 10% O2 under high relative humidity for 48 h. The objective of this study was to test CATT against P. pallidus in North America at a scale that can be easily used on-farms by strawberry growers. Two greenhouse experiments were conducted where infested trayplants were treated with CATT or the acaricide abamectin, and P. pallidus number were compared to control plants. Plants were destructively sampled after 4 weeks, and CATT reduced mobile forms of P. pallidus by 99.9% in both experiments. Abamectin used in the first experiment had an efficacy of 95.5%. Our findings suggest that CATT is effective at nearly eliminating P. pallidus from strawberry planting material, and its application could reduce risks of field infestations and the need for multiple acaricide applications. While our results are encouraging, additional research is needed to assess the effects of CATT on strawberry plant survival, growth, and fruit production.

Beneficial effects of ivabradine in patients with heart failure, low ejection fraction, and heart rate above 77 b.p.m.

AIMS: Ivabradine has been approved in heart failure with reduced ejection fraction (HFrEF) and elevated heart rate despite guideline-directed medical therapy (GDMT) to reduce cardiovascular (CV) death and hospitalization for worsening HF. The median value of 77 b.p.m. is the lower bound selected for the regulatory approval in Canada, South Africa, and Australia. Patient-reported outcomes (PROs) including symptoms, quality of life, and global assessment are considered of major interest in the global plan of care of patients with HF. However, the specific impact of GDMT, and specifically ivabradine, on PRO remains poorly studied. In the subgroup of patients from the Systolic Heart failure treatment with the If inhibitor ivabradine Trial (SHIFT) who had heart rate above the median of 77 b.p.m. (pre-specified analysis) and for whom the potential for improvement was expected to be larger, we aimed (i) to evaluate the effects of ivabradine on PRO (symptoms, quality of life, and global assessment); (ii) to consolidate the effects of ivabradine on the primary composite endpoint of CV death and hospitalization for HF; and (iii) to reassess the effects of ivabradine on left ventricular (LV) remodelling. METHODS AND RESULTS: Comparisons were made according to therapy, and proportional hazards models (adjusted for baseline beta-blocker therapy) were used to estimate the association between ivabradine and various outcomes. In SHIFT, n = 3357 (51.6%) patients had a baseline heart rate > 77 b.p.m. After a median follow-up of 22.9 months (inter-quartile range 18-28 months), ivabradine on top of GDMT improved symptoms (28% vs. 23% improvement in New York Heart Association functional class, P = 0.0003), quality of life (5.3 vs. 2.2 improvement in Kansas City Cardiomyopathy Questionnaire overall summary score, P = 0.005), and global assessment [from both patient (improved in 72.3%) and physician (improved in 61.0%) perspectives] significantly more than did placebo (both P < 0.0001). Ivabradine induced a 25% reduction in the combined endpoint of CV death and hospitalization for HF (hazard ratio 0.75; P < 0.0001), which translates into a number of patients needed to be treated for 1 year of 17. Patients under ivabradine treatment demonstrated a significant reduction in LV dimensions when reassessed at 8 months (P < 0.05). CONCLUSIONS: In patients with chronic HFrEF, sinus rhythm, and a heart rate > 77 b.p.m. while on GDMT, the present analysis brings novel insights into the role of ivabradine in improving the management of HFrEF, particularly with regard to PRO (ISRCTN70429960).

Vasopressin type 2 receptor V88M mutation: molecular basis of partial and complete nephrogenic diabetes insipidus.

BACKGROUND/AIMS: Mutations in the type 2 vasopressin receptor gene (AVPR2) underlie X-linked recessive nephrogenic diabetes insipidus (NDI). Here, we report on a family with a mutation in AVPR2, c.262G>A (p.V88M). This recurrently identified mutation was previously shown to abolish AVPR2 function, yet in some affected members, urine osmolalities of up to 570 mosm/kg were observed. We detail the variable clinical phenotype and investigate its molecular basis. METHODS: Retrospective analysis of clinical data and in vitro assessment of wild-type and V88M-mutant receptors. RESULTS: Clinical data were available on 6 patients. Four of these demonstrated a substantial increase in urinary concentration after 1-desamino[8-D-arginine] vasopressin, consistent with partial NDI, while 2 did not respond. In vitro analysis revealed a reduced cell surface expression and decreased binding affinity for arginine-vasopressin of the mutant receptor, leading to blunted signaling activity. Treatment with the pharmacological chaperone SR121463 enhanced cell surface expression. CONCLUSION: The V88M mutation is associated with phenotypical diversity, which may be explained by the fact that both the expression level and the hormone-binding affinity are affected by the mutation. Our results provide a rational basis for treatment trials with vasopressin analogues in combination with pharmacologic chaperones in patients with this recurrently identified mutation.

Mutual antagonistic relationship between prostaglandin E(2) and IFN-gamma: Implications for rheumatoid arthritis.

Prostaglandin E(2) (PGE(2)) is a major mediator of inflammation and is present at high concentrations in the synovial fluid of rheumatoid arthritis (RA) patients. PGE(2), acting through the EP4 receptor, has both pro- and anti-inflammatory roles in vivo. To shed light on this dual role of PGE(2), we investigated its effects in whole blood and in primary human fibroblast-like synoviocytes (FLS). Gene expression analysis in human leukocytes, confirmed at the protein level, revealed an EP4-dependent inhibition of the expression of genes involved in the IFN-gamma-activation pathway, including IFN-gamma itself. This effect of the PGE(2)/EP4 axis on IFN-gamma is a reciprocal phenomenon since IFN-gamma blocks PGE(2) release and blocks EP receptor expression. The mutually antagonistic relationship between IFN-gamma and PGE(2) extends to downstream cytokine and chemokine release; PGE(2) counters the effects of IFN-gamma, on the release of IP-10, IL-8, TNF-alpha and IL-1beta. To gain further insight into IFN-gamma-mediated cellular events in RA, we assessed the effects of IFN-gamma on gene expression in FLS. We observed an IFN-gamma-dependent up-regulation of macrophage-attracting chemokines, and down-regulation of metalloprotease expression. These results suggest the existence of a mutually antagonistic relationship between PGE(2) and IFN-gamma, which may represent a fundamental mechanism of immune control in diseases such as RA.

Adenovirus IL-13-induced airway disease in mice: a corticosteroid-resistant model of severe asthma.

Interleukin 13 (IL-13) is considered to be a key driver of the development of airway allergic inflammation and remodeling leading to airway hyperresponsiveness (AHR). How precisely IL-13 leads to the development of airway inflammation, AHR, and mucus production is not fully understood. In order to identify key mediators downstream of IL-13, we administered adenovirus IL-13 to specifically induce IL-13-dependent inflammation in the lungs of mice. This approach was shown to induce cardinal features of lung disease, specifically airway inflammation, elevated cytokines, AHR, and mucus secretion. Notably, the model is resistant to corticosteroid treatment and is characterized by marked neutrophilia, two hallmarks of more severe forms of asthma. To identify IL-13-dependent mediators, we performed a limited-scale two-dimensional SDS-PAGE proteomic analysis and identified proteins significantly modulated in this model. Intriguingly, several identified proteins were unique to this model, whereas others correlated with those modulated in a mouse ovalbumin-induced pulmonary inflammation model. We corroborated this approach by illustrating that proteomic analysis can identify known pathways/mediators downstream of IL-13. Thus, we have characterized a murine adenovirus IL-13 lung model that recapitulates specific disease traits observed in human asthma, and have exploited this model to identify effectors downstream of IL-13. Collectively, these findings will enable a broader appreciation of IL-13 and its impact on disease pathways in the lung.

Expression and function of NPSR1/GPRA in the lung before and after induction of asthma-like disease.

A genetic contribution to asthma susceptibility is well recognized, and linkage studies have identified a large number of genes associated with asthma pathogenesis. Recently, a locus encoding a seven-transmembrane protein was shown to be associated with asthma in founder populations. The expression of the protein GPRA (G protein-coupled receptor for asthma susceptibility) in human airway epithelia and smooth muscle, and its increased expression in a mouse model of asthma, suggested that a gain-of-function mutation in this gene increased the disease risk. However, we report here that the development of allergic lung disease in GPRA-deficient mice is unaltered. A possible explanation for this finding became apparent upon reexamination of the expression of this gene. In contrast to initial studies, our analyses failed to detect expression of GPRA in human lung tissue or in mice with allergic lung disease. We identify a single parameter that distinguishes GPRA-deficient and wild-type mice. Whereas the change in airway resistance in response to methacholine was identical in control and GPRA-deficient mice, the mutant animals showed an attenuated response to thromboxane, a cholinergic receptor-dependent bronchoconstricting agent. Together, our studies fail to support a direct contribution of GPRA to asthma pathogenesis. However, our data suggest that GPRA may contribute to the asthmatic phenotype by altering the activity of other pathways, such as neurally mediated mechanisms, that contribute to disease. This interpretation is supported by high levels of GPRA expression in the brain and its recent identification as the neuropeptide S receptor.

Structure-function relationships in the neuropeptide S receptor: molecular consequences of the asthma-associated mutation N107I.

Neuropeptide S (NPS) and its receptor (NPSR) are thought to have a role in asthma pathogenesis; a number of single nucleotide polymorphisms within NPSR have been shown to be associated with an increased prevalance of asthma. One such single nucleotide polymorphism leads to the missense mutation N107I, which results in an increase in the potency of NPS for NPSR. To gain insight into structure-function relationships within NPS and NPSR, we first carried out a limited structural characterization of NPS and subjected the peptide to extensive mutagenesis studies. Our results show that the NH(2)-terminal third of NPS, in particular residues Phe-2, Arg-3, Asn-4, and Val-6, are necessary and sufficient for activation of NPSR. Furthermore, part of a nascent helix within the peptide, spanning residues 5 through 13, acts as a regulatory region that inhibits receptor activation. Notably, this inhibition is absent in the asthma-linked N107I variant of NPSR, suggesting that residue 107 interacts with the aforementioned regulatory region of NPS. Whereas this interaction may be at the root of the increase in potency associated with the N107I variant, we show here that the mutation also causes an increase in cell-surface expression of the mutant receptor, leading to a concomitant increase in the maximal efficacy (E(max)) of NPS. Our results identify the key residues of NPS involved in NPSR activation and suggest a molecular basis for the functional effects of the N107I mutation and for its putative pathophysiological link with asthma.

Pharmacologic chaperones as a potential treatment for X-linked nephrogenic diabetes insipidus.

In many mendelian diseases, some mutations result in the synthesis of misfolded proteins that cannot reach a transport-competent conformation. In X-linked nephrogenic diabetes insipidus, most of the mutant vasopressin 2 (V2) receptors are trapped in the endoplasmic reticulum and degraded. They are unable to reach the plasma membrane and promote water reabsorption through the principal cells of the collecting ducts. Herein is reported two types of experiments: In vivo studies to assess clinically a short-term treatment with a nonpeptide V1a receptor antagonist (SR49059) and in vitro studies in cultured cell systems. In patients, SR49059 decreased 24- h urine volume (11.9 +/- 2.3 to 8.2 +/- 2.0 L; P = 0.005) and water intake (10.7 +/- 1.9 to 7.2 +/- 1.6 L; P < 0.05). Maximum increase in urine osmolality was observed on day 3 (98 +/- 22 to 170 +/- 52 mOsm/kg; P = 0.05). Sodium, potassium, and creatinine excretions and plasma sodium were constant throughout the study. In vitro studies indicate that the nonpeptide V1a receptor antagonist SR49059 and the V1a/V2 receptor antagonist YM087 (Conivaptan) rescued cell surface expression and function of mutant V2 receptors. Mutant V2 receptors with nonsense mutations were not affected by the treatment. Misfolded V2 receptor mutants were rescued in vitro and also in vivo by nonpeptide antagonists. This therapeutic approach could be applied to the treatment of several hereditary diseases that result from errors in protein folding and kinesis.

Pharmacological chaperone action on G-protein-coupled receptors.

An increasing number of genetic diseases are found to result from mutations that lead to retention of the affected proteins in the endoplasmic reticulum, where they are recognized as misfolded by the quality control system. Several of these conformational diseases involve mutations in G-protein-coupled receptors. Recent studies demonstrated that pharmacologically selective compounds, termed pharmacological chaperones, can stabilize the misfolded receptors, facilitating their export from the endoplasmic reticulum to the plasma membrane, where they can be active. Such functional rescue suggests that pharmacological chaperones could represent novel therapeutic agents for the treatment of conformational diseases. Although only a few examples are currently available, the observation that pharmacological chaperones can also favour the folding of wild-type G-protein-coupled receptors indicates that these compounds could have wide applications.

Pharmacological chaperones: potential treatment for conformational diseases.

Increasing numbers of inherited diseases are found to result from mutations that lead to misfolded proteins. In many cases, the changes in conformation are relatively modest and the function of the protein would not be predicted to be affected. Yet, these proteins are recognized as "misfolded" and degraded prematurely. Recently, small molecules known as chemical and pharmacological chaperones were found to stabilize such mutant proteins and facilitate their trafficking to their site of action. Here, we review the recent published evidence suggesting that pharmacological chaperones represent promising avenues for the treatment of endocrine and metabolic diseases such as hyperinsulinemic hypoglycemia, hypogonadotropic hypogonadism and nephrogenic diabetes insipidus, and might become a general therapeutic strategy for the treatment of conformational diseases.

Functional rescue of the constitutively internalized V2 vasopressin receptor mutant R137H by the pharmacological chaperone action of SR49059.

In most cases, nephrogenic diabetes insipidus results from mutations in the V2 vasopressin receptor (V2R) gene that cause intracellular retention of improperly folded receptors. We previously reported that cell permeable V2R antagonists act as pharmacological chaperones that rescue folding, trafficking, and function of several V2R mutants. More recently, the vasopressin antagonist, SR49059, was found to be therapeutically active in nephrogenic diabetes insipidus patients. Three of the patients with positive responses harbored the mutation R137H, previously reported to lead to constitutive endocytosis. This raises the possibility that, instead of acting as a pharmacological chaperone by favoring proper maturation of the receptors, SR49059 could mediate its action on R137H V2R by preventing its endocytosis. Here we report that the beta-arrestin-mediated constitutive endocytosis of R137H V2R is not affected by SR49059, indicating that the functional rescue observed does not result from a stabilization of the receptor at the cell surface. Moreover, metabolic labeling revealed that R137H V2R is also poorly processed to the mature form. SR49059 treatment significantly improved its maturation and cell surface targeting, indicating that the functional rescue of R137H V2Rs results from the pharmacological chaperone action of the antagonist.