A novel CCR5-specific pharmacodynamic assay in whole blood using phosphoflow cytometry highlights different ligand-dependent responses but similar properties of antagonists in CD8+ and CD4+ T lymphocytes

Chemokine CC motif receptor (CCR) 5 is a major drug target for both inflammation and virology indications. The primary function of CCR5 is to mediate the trafficking of CCR5-expressing lymphocytes to any of the CCR5 ligands, which are often increased during inflammatory responses. In addition, CCR5 is a coreceptor for HIV, mediating R5 tropic HIV infection of CCR5-expressing CD4 T cells. We report the use of a novel method to assay the pharmacodynamic (PD) properties of small-molecule and antibody inhibitors of CCR5 ligand-induced activation by measuring phosphorylation of serine residue 349 in the cytoplasmic tail of human CCR5 using phosphoflow cytometry in whole blood. This assay is highly specific and measures CCR5 phosphorylation in both CD8(+) and CD4(+) T cells and allows the calculation of inhibitor IC(50) values from both lymphocyte subsets in the presence of CCR5 antagonists. In addition, this assay is cross-reactive to nonhuman primates and allows PD analysis in whole blood from rhesus and cynomolgus macaque. Using this assay, we identified different ligand-dependent response properties between CD8(+) and CD4(+) T cells, although CCR5 antagonists behave with similar properties against both cell types. The use of this assay may be of particular benefit to monitor PD effects of CCR5 inhibitors during drug development, preclinical in vivo studies, and in patients currently being treated for HIV or CCR5-mediated inflammatory diseases with CCR5 inhibitors. Similar phosphoflow approaches to other GPCR targets on circulating lymphocytes may prove to be the most reliable PD assay for preclinical and potentially clinical development.     

The Costs of Respiratory Illnesses Arising from Florida Gulf Coast Karenia brevis Blooms

Background

Algal blooms of Karenia brevis, a harmful marine algae, occur almost annually off the west coast of Florida. At high concentrations, K. brevis blooms can cause harm through the release of potent toxins, known as brevetoxins, to the atmosphere. Epidemiologic studies suggest that aerosolized brevetoxins are linked to respiratory illnesses in humans.

Objectives

We hypothesized a relationship between K. brevis blooms and respiratory illness visits to hospital emergency departments (EDs) while controlling for environmental factors, disease, and tourism. We sought to use this relationship to estimate the costs of illness associated with aerosolized brevetoxins.

Methods

We developed a statistical exposure–response model to express hypotheses about the relationship between respiratory illnesses and bloom events. We estimated the model with data on ED visits, K. brevis cell densities, and measures of pollen, pollutants, respiratory disease, and intra-annual population changes.

Results

We found that lagged K. brevis cell counts, low air temperatures, influenza outbreaks, high pollen counts, and tourist visits helped explain the number of respiratory-specific ED diagnoses. The capitalized estimated marginal costs of illness for ED respiratory illnesses associated with K. brevis blooms in Sarasota County, Florida, alone ranged from $0.5 to $4 million, depending on bloom severity.

Conclusions

Blooms of K. brevis lead to significant economic impacts. The costs of illness of ED visits are a conservative estimate of the total economic impacts. It will become increasingly necessary to understand the scale of the economic losses associated with K. brevis blooms to make rational choices about appropriate mitigation.     

Elastic fiber depletion in the supporting ligaments of the gastroesophageal junction: a structural basis for the development of hiatal hernia

BACKGROUND: The position of the gastroesophageal junction is maintained by a complex of fibroelastic ligaments. The purpose of this study was to characterize and compare the histology of these ligaments in patients with gastroesophageal reflux disease (GERD) and hiatal hernia (HH) versus GERD alone, with emphasis on the elastin morphology. STUDY DESIGN: Thirteen patients were examined at the time of laparoscopic fundoplication for symptomatic GERD; nine had no significant HH and four had large diaphragmatic hernias (GERD/HH). Tissue biopsies were obtained from the gastrohepatic ligament (GHL, n=5 and n=3, GERD and GERD/HH, respectively), the phrenoesophageal ligament (n=7 and n=4, respectively), and the gastrophrenic ligament (n=6 and n=4, respectively). Sections of fixed tissue were stained with hematoxylin and eosin, Masson's trichrome, and resorcin-fuchsin for analysis of elastic fibers by light microscopy, and elastin area was quantified and expressed as a percentage of the imaged tissue. RESULTS: Elastin and collagen fibers were prominent in all ligaments in patients with GERD alone. In patients with GERD/HH, there was fragmentation and distortion of elastin in the phrenoesophageal ligament and gastrohepatic ligament, and to a lesser degree, in the gastrophrenic ligament. Compared with patients with GERD alone, the presence of hiatal hernia was associated with a reduction in elastin area by more than 50% in the phrenoesophageal ligament ([mean +/- SEM] 31.0%+/-3.3% versus 15.1%+/-1.3%, p < 0.01) and gastrohepatic ligament (26.9% +/- 0.5% versus 12.5%+/-0.1%, p < 0.008). There was no decrease with respect to elastin in the gastrophrenic ligament. CONCLUSIONS: The periesophageal ligaments in patients with GERD are characterized by prominent elastic fibers. In contrast, GERD/HH is associated with depletion of elastic fibers in two of three ligaments supporting the gastroesophageal junction. Elastic fiber depletion in the periesophageal ligaments thereby provides a structural basis for the development of HH. It remains unclear if this represents a primary (etiologic) alteration or if it is a secondary phenomenon.     

Design, feasibility, and acceptability of an intervention using personal digital assistant-based self-monitoring in managing type 2 diabetes

BackgroundThe information processing demands associated with behavioral self-management of diabetes are extensive. Pairing personal digital assistant (PDA)-based self-monitoring with a behavioral self-management intervention may improve adherence and patient outcomes.MethodsENHANCE is a randomized controlled trial to test an intervention designed to improve regimen adherence in adults with type 2 diabetes. The intervention, based on Social Cognitive Theory (SCT), is paired with PDA-based self-monitoring. In this paper we describe the: (a) manner in which PDA-based self-monitoring is integrated within the SCT-based intervention, (b) feasibility and acceptability of PDA-based dietary self-monitoring, and (c) issues encountered in teaching participants to self-monitor using a PDA.ResultsDuring the first 30 months of this 5-year study, 232 participants were screened and 151 were randomized. To date, 6 cohorts have completed the study. The retention rate is 85% (n = 129). Of those randomized to the intervention (n = 74) and completing the study (n = 61), 88% reported understanding the usefulness of PDA-monitoring, 85% reported ease in entering foods into the device, 70% reported ease in interpreting feedback graphs, and 82% indicated that they would continue to use the PDA for self-monitoring after the study concluded. Assuming 3 meals per day, participants entered an average of 58% of their meals in their PDA, and 43% were entered assuming 4 meals per day. If we eliminate from the analysis those individuals who entered less than 10% of their expected meals (n = 12), the average rate of self-monitoring was 69% assuming 3 meals per day, and 52% assuming 4 meals per day.ConclusionsPDA-based dietary monitoring is perceived by participants to be useful and acceptable. The approach used to instruct participants in use of the PDA and lessons learned are discussed. PDA technology shows promise as a tool for assisting those with type 2 diabetes in their efforts to manage their disease.     

Melanocortin receptors as targets in the treatment of obesity

The central melanocortin system plays a critical role in energy homeostasis. It is well established that melanocortin-containing neurons are nutritionally regulated and that genetic alterations in the melanocortin system produce profound effects on food intake, energy expenditure, and body weight. Within the brain, melanocortin-producing neurons originate in the arcuate nucleus of the hypothalamus (ARC) and the nucleus of the solitary tract (NTS) in the brainstem and project to various nuclei modulating energy balance. A large body of pharmacological and genetic evidence implicates the central melanocortin 4 receptors (MC4Rs) in the effects of melanocortin peptides on ingestive behaviour, energy expenditure, and body weight. Preclinical studies with endogenous and synthetic melanocortin ligands demonstrate that they produce potent effects on food intake and energy expenditure. Clinical studies thus far have been somewhat less successful and have been hampered by the induction of side effects, which present obstacles to the development of successful therapeutic agents. However, various promising strategies are being pursued to overcome these limitations, including the synthesis of more selective and potent melanocortin analogs.     

Effect of extended-term estrogen on voiding in a postpartum ovariectomized rat model

Introduction

We tested the hypothesis that extended-term (5-week) estrogen therapy would negatively impact voiding function in a postpartum, ovariectomized rat model.

Methods

Immediately after delivery, 30 primiparous Sprague–Dawley rats underwent intravaginal balloon dilation, followed by ovariectomy 1 week later. Cystometry at postpartum week 2 determined normal or abnormal voiding patterns. After randomization, one-half the normal and abnormal voiding rats received 5 weeks of estrogen therapy, while the remainder received placebo. Estrogen effect was determined by repeat cystometry and immunohistochemical analysis of the urethra and vagina.

Results

Abnormal voiding increased from 60.0% to 73.3% in the estrogen- treated group and declined from 60% to 33% for the placebo group. Rats were then divided into 4 groups for comparison: normal voiding versus placebo (group 1), abnormal voiding versus placebo (group 2), normal voiding versus estrogen (group 3) and abnormal voiding versus estrogen (group 4). Bladder capacity, leak point pressure and maximum voiding pressure were most depressed in group 4. Estrogen treatment was associated with a significant downregulation of α_1A and α_1D-adrenoceptors in the urethral submucosa but an upregulation of nNOS in the urethral smooth muscle.

Conclusion

Extended-term estrogen therapy in a rat model of simulated birth trauma and ovariectomy resulted in a higher rate of incontinence. Immunohistochemical examination demonstrated significant downregulation of urethral α_1A- and α_1D-adrenoceptors and upregulation of neuronal nitric oxide synthase (nNOS) in the urethra of estrogen-treated groups. These studies question the use of hormone replacement therapy in the treatment of postmenopausal incontinence.     

Identification of Ras-related nuclear protein, targeting protein for xenopus kinesin-like protein 2, and stearoyl-CoA desaturase 1 as promising cancer targets from an RNAi-based screen

To identify new candidate cancer drug targets, we used RNAi as a tool to functionally evaluate genes that play a role in maintaining human tumor cell survival. We screened a small interfering RNA (siRNA) library directed against approximately 3,700 individual genes to assess the ability of siRNAs to induce cell death in an in vitro cell cytotoxicity assay. We found that siRNAs specifically targeting ras-related nuclear protein (Ran), targeting protein for Xenopus kinesin-like protein 2 (TPX2), and stearoyl-CoA desaturase 1 (SCD1), significantly reduced the survival of multiple human tumor cell lines. Further target validation studies revealed that treatment with Ran and TPX2 siRNAs differentially reduced the survival of activated K-Ras-transformed cells compared with their normal isogenic counterparts in which the mutant K-Ras gene had been disrupted (DKS-8). Knockdown of Ran and TPX2 in activated mutant K-Ras cells selectively induced S-phase arrest or transient G(2)-M arrest phenotypes, respectively, that preceded apoptotic cell death. Given our observations that Ran and TPX2 depletion preferentially reduces the survival of activated K-Ras-transformed cells, these two proteins may serve as useful anticancer targets in tumors expressing the activated K-Ras oncogene.     

Serotonin gene polymorphisms and bipolar I disorder: Focus on the serotonin transporter

The pathogenesis of bipolar disorder may involve, at least in part, aberrations in serotonergic neurotransmission. Hence, serotonergic genes are attractive targets for association studies of bipolar disorder. We have reviewed the literature in this field. It is difficult to synthesize results as only one polymorphism per gene was typically investigated in relatively small samples. Nevertheless, suggestive associations are available for the 5HT2A receptor and the serotonin transporter genes. With the availability of extensive polymorphism data and high throughput genotyping techniques, comprehensive evaluation of these genes using adequately powered samples is warranted. We also report on our investigations of the serotonin transporter, SLC6A4 (17q11.1‐q12). An insertion/deletion polymorphism (5HTTLPR) in the promoter region of this gene has been investigated intensively. However, the results have been inconsistent. We reasoned that other polymorphism/s may contribute to the associations and the inconsistencies may be due to variations in linkage disequilibrium (LD) patterns between samples. Therefore, we conducted LD analyses, as well as association and linkage using 12 polymorphisms, including 5HTTLPR. We evaluated two samples. The first sample consisted of 135 US Caucasian nuclear families having a proband with bipolar I disorder (BDI, DSM IV criteria) and available parents. For case‐control analyses, the patients from these families were compared with cord blood samples from local Caucasian live births (n = 182). Our second, independent sample was recruited through the Systematic Treatment Enhancement Program for Bipolar Disorder (STEP‐BD, 545 cases, 548 controls). No significant associations were detected at the individual polymorphism or haplotype level using the case‐control or family‐based analyses. Our analyses do not support association between SLC6A4 and BDI families. Further studies using sub‐groups of BDI are worthwhile.