Expression of the PGE2 receptor, EP4, is up-regulated during colorectal carcinogenesis. However the mechanism leading to deregulation of the EP4 receptor is not known. The present study was conducted to investigate the regulation of EP4 receptor by miRNAs.
We analyzed 26 colon cancers (i.e. 15 adenocarcinomas and 9 adenomas) and 16 normal colon specimens for EP4 receptor expression by immunohistochemistry. A bioinformatics approached identified putative microRNA binding sites with the 3′-UTR of the EP4 receptor. Both colon cancer cell lines and tumor specimens were analyzed for miR-101 and EP4 expression by qRT-PCR and Western analysis respectively and simultaneously in situ hybridizations was used to confirm our results. In vitro and in vivo assays were used to confirm our clinical findings.
We observed an inverse correlation between the levels of miR-101 and EP4 receptor protein. Transfection of LS174T cells with miR-101 significantly suppressed a luciferase reporter containing the EP4 receptor-3′-UTR. In contrast, a mutant EP4 receptor-3′-UTR construct was unaffected. Ectopic expression of miR-101 markedly reduced cell proliferation and motility. Co-transfection of EP4 receptor could rescue colon cancer cells from the tumor suppressive effects of miR- 101. Moreover, the pharmacologic inhibition of EP4 receptor signaling or silencing of EP4 receptor phenocopied the effect of miR-101. This is the first study to show that the EP4 receptor is negatively regulated by miR-101.
These data provide new insights in the modulation of EP-4 receptor expression at the post-transcriptional level by miR-101 and suggests therapeutic strategies against miR-101 targets may be warranted.
colorectal cancer; EP4; miR-101; post-transcriptional regulation
Acidosis is a powerful vasodilator signal in the brain circulation. However, the mechanisms by which this response occurs are not well understood, particularly in the cerebral microcirculation. One important mechanism to dilate cerebral (pial) arteries is by activation of large-conductance, calcium-sensitive potassium (BKCa) channels by local Ca2+ signals (Ca2+ sparks) through ryanodine receptors (RyRs). However, the role of this pathway in the brain microcirculation is not known.
The objectives of this study were to determine the mechanism by which acidosis dilates brain parenchymal arterioles (PAs) and to elucidate the roles of RyRs and BKCa channels in this response.
Methods and Results
Internal diameter and vascular smooth muscle cell (VSMC) Ca2+ signals were measured in isolated pressurized murine PAs, using imaging techniques. In physiological pH (7.4), VSMCs exhibited primarily RyR-dependent Ca2+ waves. Reducing external pH from 7.4 to 7.0 in both normocapnic and hypercapnic conditions decreased Ca2+ wave activity, and dramatically increased Ca2+ spark activity. Acidic pH caused a dilation of PAs which was inhibited by about 60% by BKCa channel or RyR blockers, in a non-additive manner. Similarly, dilator responses to acidosis were reduced by nearly 60% in arterioles from BKCa channel knockout mice. Dilations induced by acidic pH were unaltered by inhibitors of KATP channels or nitric oxide synthase.
These results support the novel concept that acidification, by converting Ca2+ waves to sparks, leads to the activation of BKCa channels to induce dilation of cerebral parenchymal arterioles.
brain parenchymal arteriole; acidosis; Ca2+ sparks; ryanodine receptor; potassium channel
Respiratory infections including atypical bacteria Mycoplasma pneumoniae (Mp) contribute to the pathobiology of asthma and chronic obstructive pulmonary disease (COPD). Mp infection mainly targets airway epithelium and activates various signaling pathways such as nuclear factor κB (NF-κB). We have shown that short palate, lung, and nasal epithelium clone 1 (SPLUNC1) serves as a novel host defense protein and is up-regulated upon Mp infection through NF-κB activation in cultured human and mouse primary airway epithelial cells. However, the in vivo role of airway epithelial NF-κB activation in host defense against Mp infection has not been investigated. In the current study, we investigated the effects of in vivo airway epithelial NF-κB activation on lung Mp clearance and its association with airway epithelial SPLUNC1 expression.
Non-antimicrobial tetracycline analog 9-t-butyl doxycycline (9-TB) was initially optimized in mouse primary tracheal epithelial cell culture, and then utilized to induce in vivo airway epithelial specific NF-κB activation in conditional NF-κB transgenic mice (CC10-CAIKKβ) with or without Mp infection. Lung Mp load and inflammation were evaluated, and airway epithelial SPLUNC1 protein was examined by immunohistochemistry. We found that 9-TB treatment in NF-κB transgene positive (Tg+), but not transgene negative (Tg−) mice significantly reduced lung Mp load. Moreover, 9-TB increased airway epithelial SPLUNC1 protein expression in NF-κB Tg+ mice.
By using the non-antimicrobial 9-TB, our study demonstrates that in vivo airway epithelial NF-κB activation promotes lung bacterial clearance, which is accompanied by increased epithelial SPLUNC1 expression.
To investigate and examine the factors associated with initiation of, and exclusive breastfeeding at hospital discharge of, late preterm (34 0/7 - 36 6/7 weeks) compared to 37 week gestation (37 0/7 - 37 6/7 week) mother and baby pairs.
A retrospective population-based cohort study using a Perinatal National Minimum Data Set and clinical medical records review, at the Royal Hobart Hospital, Tasmania, Australia in 2006.
Late preterm and 37 week gestation infants had low rates of initiation of breastfeeding within one hour of birth, 31 (21.1%) and 61 (41.5%) respectively. After multiple regression analysis, late preterm infants were less likely to initiate breastfeeding within one hour of birth (OR 0.3 95% CI 0.1, 0.7 p = 0.009) and were less likely to be discharged exclusively breastfeeding from hospital (OR 0.4 95% CI 0.1, 1.0 p = 0.04) compared to 37 week gestation infants.
A late preterm birth is predictive of breastfeeding failure, with late preterm infants at greater risk of not initiating breastfeeding and/or exclusively breastfeeding at hospital discharge, compared with those infants born at 37 weeks gestation. Stratifying breastfeeding outcomes by gestational age groups may help to identify those sub-populations at greatest risk of premature cessation of breastfeeding.
Exclusive breastfeeding; Initiation; Late preterm; Infant
Hepatitis B virus (HBV) infection is an increasingly important cause of morbidity and mortality in HIV-infected adults. This study aimed to determine the prevalence and incidence of HBV in the UK CHIC Study, a multicentre observational cohort.
Methods and Findings
12 HIV treatment centres were included. Of 37,331 patients, 27,450 had at least one test (HBsAg, anti-HBs or anti-HBc) result post-1996 available. 16,043 were white, 8,130 black and 3,277 other ethnicity. Route of exposure was homosexual sex 15,223 males, heterosexual sex 3,258 males and 5,384 females, injecting drug use 862 and other 2,723. The main outcome measures used were the cumulative prevalence and the incidence of HBV coinfection. HBV susceptible patients were followed up until HBsAg and/or anti-HBc seroconversion incident infection, evidence of vaccination or last visit. Poisson regression was used to determine associated factors. 25,973 had at least one HBsAg test result. Participants with HBsAg results were typically MSM (57%) and white (59%) (similar to the cohort as a whole). The cumulative prevalence of detectable HBsAg was 6.9% (6.6 to 7.2%). Among the 3,379 initially HBV-susceptible patients, the incidence of HBV infection was 1.7 (1.5 to 1.9)/100 person-years. Factors associated with incident infection were older age and IDU. The main limitation of the study was that 30% of participants did not have any HBsAg results available. However baseline characteristics of those with results did not differ from those of the whole cohort. Efforts are on-going to improve data collection.
The prevalence of HBV in UK CHIC is in line with estimates from other studies and low by international standards. Incident infection continued to occur even after entry to the cohort, emphasising the need to ensure early vaccination.
To determine the level of agreement between a ‘conventional’ Ankle-Brachial Index (ABI) measurement (using Doppler and mercury sphygmomanometer taken by a research nurse) and a ‘pragmatic’ ABI measure (using an oscillometric device taken by a practice nurse) in primary care. To ascertain the utility of a pragmatic ABI measure for the diagnosis of peripheral arterial disease (PAD) in primary care.
Cross-sectional validation and diagnostic accuracy study. Descriptive analyses were used to investigate the agreement between the two procedures using the Bland and Altman method to determine whether the correlation between ABI readings varied systematically. Diagnostic accuracy was assessed via sensitivity, specificity, accuracy, likelihood ratios, positive and negative predictive values, with ABI readings dichotomised and Receiver Operating Curve analysis using both univariable and multivariable logistic regression.
Primary care in metropolitan and rural Victoria, Australia between October 2009 and November 2010.
250 persons with cardiovascular disease (CVD) or at high risk (three or more risk factors) of CVD.
Despite a strong association between the two method's measurements of ABI there was poor agreement with 95% of readings within ±0.4 of the 0.9 ABI cut point. The multivariable C statistic of diagnosis of PAD was 0.89. Other diagnostic measures were sensitivity 62%, specificity 92%, positive predictive value 67%, negative predictive value 90%, accuracy 85%, positive likelihood ratio 7.3 and the negative likelihood ratio 0.42.
Oscillometric ABI measures by primary care nurses on a population with a 22% prevalence of PAD lacked sufficient agreement with conventional measures to be recommended for routine diagnosis of PAD. This pragmatic method may however be used as a screening tool high-risk and overt CVD patients in primary care as it can reliably exclude the condition.
Primary Care; Vascular Medicine; Public Health
The aim of the study is to describe the work pattern of personal care workers (PCWs) in nursing homes. This knowledge is important for staff performance appraisal, task allocation and scheduling. It will also support funding allocation based on activities.
A time-motion study was conducted in 2010 at two Australian nursing homes. The observation at Site 1 was between the hours of 7:00 and 14:00 or 15:00 for 14 days. One PCW was observed on each day. The observation at Site 2 was from 10:00 to 17:00 for 16 days. One PCW working on a morning shift and another one working on an afternoon shift were observed on each day. Fifty-eight work activities done by PCWs were grouped into eight categories. Activity time, frequency, duration and the switch between two consecutive activities were used as measurements to describe the work pattern.
Personal care workers spent about 70.0% of their time on four types of activities consistently at both sites: direct care (30.7%), indirect care (17.6%), infection control (6.4%) and staff break (15.2%). Oral communication was the most frequently observed activity. It could occur independently or concurrently with other activities. At Site 2, PCWs spent significantly more time than their counterparts at Site 1 on oral communication (Site 1: 47.3% vs. Site 2: 63.5%, P = 0.003), transit (Site 1: 3.4% vs. Site 2: 5.5%, P < 0.001) and others (Site 1: 0.5% vs. Site 2: 1.8%, P < 0.001). They spent less time on documentation (Site 1: 4.1% vs. Site 2: 2.3%, P < 0.001). More than two-thirds of the observed activities had a very short duration (1 minute or less). Personal care workers frequently switched within or between oral communication, direct and indirect care activities.
At both nursing homes, direct care, indirect care, infection control and staff break occupied the major part of a PCW’s work, however oral communication was the most time consuming activity. Personal care workers frequently switched between activities, suggesting that looking after the elderly in nursing homes is a busy and demanding job.
Maraviroc (MVC) is the first licensed antiretroviral therapeutic agent to target a host cell surface molecule, and successful HIV-1 entry blockade by this C-C chemokine receptor type 5 (CCR5)-antagonist potentiates immunomodulation. We hypothesized that MVC intensification impacts immunization responses, T-cell phenotype, function and delayed type hypersensitivity (DTH) in HIV-1+ subjects. A 24-wk, double-blinded, placebo-controlled study of the addition of MVC to suppressive antiretroviral therapy in HIV-1+ persons was performed. Subjects received DTH tests, intramuscular tetanus, meningococcal and oral cholera immunizations. Antibody titers, T-cell function and phenotype were assessed. Of 157 patients referred, 47 were randomized 1:1; MVC:placebo. MVC enhanced meningococcal neo-immunization, blunted cholera response and expedited lymphoproliferation to tetanus boost, without affecting recall humoral response. Anti-HIV-1 group-specific antigen (Gag) and tetanus toxoid (TTox) function improved significantly, HIV-1-associated CD8 T-cell skewing normalized, and the percentage of late-stage and major histocompatibility complex (MHC) class II expressing CD4 T-cells increased. Activated CD4+ CD38+ human leukocyte antigen (HLA)-DR+ T-cells declined, and costimulation shifted to coinhibition. DTH was unchanged. Maraviroc intensification, through antagonism of the cell surface molecule CCR5, favorably influences immune profiles of HIV-1+ patients, supporting its immunomodulatory use in HIV-1 infection and potentially in other immunologically relevant settings.
Adoption and maintenance of healthy behaviours is pivotal to chronic disease self-management as this influences disease progression and impact. This qualitative study investigated health behaviour changes adopted by participants with moderate or severe chronic obstructive pulmonary disease (COPD) recruited to a randomised controlled study of telephone-delivered health-mentoring.
Community nurses trained as health-mentors used a patient-centred approach with COPD patients recruited in general practice to facilitate behaviour change, using a framework of health behaviours; ‘SNAPPS’ Smoking, Nutrition, Alcohol, Physical activity, Psychosocial well-being, and Symptom management, through regular phone calls over 12 months. Semi-structured interviews in a purposive sample sought feedback on mentoring and behaviour changes adopted. Interviews were analysed using iterative thematic and interpretative content approaches by two investigators.
Of 90 participants allocated to health-mentoring, 65 (72%) were invited for interview at 12-month follow up. The 44 interviewees, 75% with moderate COPD, had a median of 13 mentor contacts over 12 months, range 5–20. Interviewed participants (n = 44, 55% male, 43% current smokers, 75% moderate COPD) were representative of the total group with a mean age 65 years while 82% had at least one additional co-morbid chronic condition. Telephone delivery was highly acceptable and enabled good rapport. Participants rated ‘being listened to by a caring health professional’ as very valuable. Three participant groups were identified by attitude to health behaviour change: 14 (32%) actively making changes; 18 (41%) open to and making some changes and 12 (27%) more resistant to change. COPD severity or current smoking status was not related to group category. Mentoring increased awareness of COPD effects, helping develop and personalise behaviour change strategies, even by those not actively making changes. Physical activity was targeted by 43 (98%) participants and smoking by 14 (74%) current smokers with 21% reporting quitting. Motivation to maintain changes was increased by mentor support.
Telephone delivery of health-mentoring is feasible and acceptable to people with COPD in primary care. Health behaviours targeted by this population, mostly with moderate disease, were mainly physical activity and smoking reduction or cessation. Health-mentoring increased motivation and assisted people to develop strategies for making and sustaining beneficial change.
Health-mentoring; Behaviour change; Primary care; Chronic obstructive pulmonary disease; Physical activity; Smoking
Calcium-sensitive potassium (KCa) channels have been shown to modulate the diameter of cerebral pial arteries; however, little is known regarding their roles in controlling cerebral parenchymal arterioles (PAs). We explored the function and cellular distribution of small-conductance (SKCa) and intermediate-conductance (IKCa) KCa channels and large-conductance KCa (BKCa) channels in endothelial cells (ECs) and smooth muscle cells (SMCs) of PAs. Both SKCa and IKCa channels conducted the outward current in isolated PA ECs (current densities, ∼20 pA/pF and ∼28 pA/pF at +40 mV, respectively), but these currents were not detected in PA SMCs. In contrast, BKCa currents were prominent in PA SMCs (∼154 pA/pF), but were undetectable in PA ECs. Pressurized PAs constricted to inhibition of SKCa (∼16%) and IKCa (∼16%) channels, but were only modestly affected by inhibition of BKCa channels (∼5%). Blockade of SKCa and IKCa channels decreased resting cortical cerebral blood flow (CBF) by ∼15%. NS309 (6,7-dichloro-1H-indole-2,3-dione3-oxime), a SKCa/IKCa channel opener, hyperpolarized PA SMCs by ∼27 mV, maximally dilated pressurized PAs, and increased CBF by ∼40%. In conclusion, these data show that SKCa and IKCa channels in ECs profoundly modulate PA tone and CBF, whereas BKCa channels in SMCs only modestly influence PA diameter.
BKCa; calcium-activated potassium channel; IKCa; parenchymal arterioles; SKCa
Spinning disk confocal laser microscopy systems can be used for observing fast events occurring in a small volume when they include a sensitive electron-multiplying CCD camera. Such a confocal system was recently used to capture the first pictures of intracellular calcium signalling within the projections of endothelial cells to the adjacent smooth muscle cells in the blood vessel wall. Detection of these calcium signals required high spatial and temporal resolution. A newly developed calcium ion (Ca2+) biosensor was also used. This exclusively expressed in the endothelium and fluoresced when Ca2+ concentrations increased during signalling. This work gives insights into blood vessel disease because Ca2+ signalling is critical for blood flow and pressure regulation.
confocal microscopy; laser spinning disk; electron-multiplying CCD camera; endothelial cells; intracellular signals; blood vessels
Maraviroc, the first approved CCR5 antagonist, demonstrated 48-week safety and virologic efficacy in CCR5-tropic HIV-infected, treatment-experienced patients; however, critical longer-term safety and durability of responses are unknown.
Two-year follow-up of 2 prospective, randomized, blinded studies of maraviroc once daily or twice daily, or placebo in treatment-experienced patients with R5-tropic HIV-1 receiving an optimized background regimen. Unblinding occurred after the week-48 visit of the last enrolled patient. Safety and virologic parameters were assessed through week 96.
One thousand forty-nine patients were randomized and received study drugs. HIV-1 RNA was <50 copies per milliliter at week 96 in 39% and 41% of patients receiving maraviroc every day or twice a day, respectively. Among patients with HIV-1 RNA <50 copies per milliliter at week 48, 81% and 87% of patients receiving maraviroc every day or twice a day, respectively, maintained this response at week 96. At week 96, median CD4+ T-cell counts increased from baseline by 89 and 113 cells per cubic millimeter with maraviroc every day and twice a day, respectively. Exposure-adjusted rates of adverse events were similar with maraviroc or placebo. No new or unexpected events were observed after week 48.
Maraviroc-containing antiretroviral regimens maintained durable responses in treatment-experienced patients with R5 HIV-1 through 96 weeks of treatment with a safety profile similar to placebo.
HIV; host-based therapies; maraviroc; long-term safety; treatment experienced
Define and identify long-term non-progressors (LTNP) and HIV controllers (HIC), and estimate time until disease progression. LTNP are HIV-1+ patients who maintain stable CD4+ T-cell counts, with no history of opportunistic infection or antiretroviral therapy (ART). HIC are a subset of LTNP who additionally have undetectable viraemia. These individuals may provide insights for prophylactic and therapeutic development. Records of HIV-1+ individuals attending Chelsea and Westminster Hospital (1988–2010), were analysed. LTNP were defined as: HIV-1+ for >7 years; ART-naïve; no history of opportunistic infection and normal, stable CD4+ T-cell counts. MIXED procedure in SAS using random intercept model identified long-term stable CD4+ T-cell counts. Survival analysis estimated time since diagnosis until disease progression. Subjects exhibiting long-term stable CD4+ T-cell counts with history below the normal range (<450 cells/µl blood) were compared to LTNP whose CD4+ T-cell count always remained normal. Within these two groups subjects with HIV-1 RNA load below limit of detection (BLD) were identified. Of 14,227 patients, 1,204 were diagnosed HIV-1+ over 7 years ago and were ART-naïve. Estimated time until disease progression for the 20% (239) whose CD4+ T-cell counts remained within the normal range, was 6.2 years (IQR: 2.0 to 9.6); significantly longer than 4.0 years (IQR: 1.0 to 7.3) for patients with historical CD4+ T-cell count below normal (Logrank chi-squared = 21.26; p<0.001). Within a subpopulation of 312 asymptomatic patients, 50 exhibited long-term stable CD4+ T-cell counts. Of these, 13 were LTNP, one of whom met HIC criteria. Of the remaining 37 patients with long-term stable low CD4+ T-cell counts, 3 controlled HIV-1 RNA load BLD. Individuals with stable, normal CD4+ T-cell counts progressed less rapidly than those with low CD4+ T-cell counts. Few LTNP and HIC identified in this and other studies, endorse the need for universal definitions to facilitate comparison.
Intracellular Ca2+ release events (‘Ca2+ sparks') and transient activation of large-conductance Ca2+-activated potassium (BK) channels represent an important vasodilator pathway in the cerebral vasculature. Considering the frequent occurrence of cerebral artery constriction after subarachnoid hemorrhage (SAH), our objective was to determine whether Ca2+ spark and BK channel activity were reduced in cerebral artery myocytes from SAH model rabbits. Using laser scanning confocal microscopy, we observed ∼50% reduction in Ca2+ spark activity, reflecting a decrease in the number of functional Ca2+ spark discharge sites. Patch-clamp electrophysiology showed a similar reduction in Ca2+ spark-induced transient BK currents, without change in BK channel density or single-channel properties. Consistent with a reduction in active Ca2+ spark sites, quantitative real-time PCR and western blotting revealed decreased expression of ryanodine receptor type 2 (RyR-2) and increased expression of the RyR-2-stabilizing protein, FKBP12.6, in the cerebral arteries from SAH animals. Furthermore, inhibitors of Ca2+ sparks (ryanodine) or BK channels (paxilline) constricted arteries from control, but not from SAH animals. This study shows that SAH-induced decreased subcellular Ca2+ signaling events disable BK channel activity, leading to cerebral artery constriction. This phenomenon may contribute to decreased cerebral blood flow and poor outcome after aneurysmal SAH.
cerebral aneurysm; FKBP12.6; potassium channels; ryanodine receptors; vascular smooth muscle; vasospasm
Prostaglandin E2 (PGE2) levels are frequently elevated in colorectal carcinomas. PGE2 is perceived via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is most relevant. PGE2/EP4-receptor interaction activates CREB via the ERK/MEK pathway. However, the downstream target genes activated by this pathway remained to be investigated.
Here, we have identified S100P (an EF-hand calcium binding protein) as a novel downstream target. We show by realtime RT-PCR that S100P mRNA levels are elevated in 14/17 (82%) colon tumor tissues as compared to paired adjacent normal colonic tissues. S100P expression is stimulated in the presence of PGE2 in a time dependent manner at mRNA and protein levels in colon, breast and pancreatic cancer cells. Pharmacological and RNAi-mediated inhibition of the EP4 receptor attenuates PGE2-dependent S100P mRNA induction. RNAi-mediated knockdown of CREB inhibits endogenous S100P expression. Furthermore, using luciferase reporter analysis and EMSA we show that mutation and/or deletion of the CRE sequence within the S100P promoter abolished PGE2-mediated transcriptional induction. Finally, we demonstrate that RNAi-mediated knockdown of S100P compromised invadopodia formation, colony growth and motility of colon cancer cells. Interestingly, endogenous knock down of S100P decreases ERK expression levels, suggesting a role for ERK in regulating S100P mediated cell growth and motility.
Together, our findings show for the first time that S100P expression is regulated by PGE2/EP4-receptor signaling and may participate in a feedback signaling that perpetuates tumor cell growth and migration. Therefore, our data suggest that dysregulated S100P expression resulting from aberrant PGE2/EP4 receptor signaling may have important consequences relevant to colon cancer pathogenesis.
Colon cancer; CREB; PGE2; EP4 receptor; ERK; S100P; RNAi
Mechanisms driving cancer-induced bone pain are poorly understood. A central factor implicated to be a key player in the process of tumorigenesis, osteoclastogenesis and nociception is p38 MAPK. We determined the role of p38 MAPK in a mouse model of breast cancer induced bone pain in which mixed osteolytic and osteoblastic remodeling occurs.
In cancer-treated mice, acute as well as chronic inhibition of p38 MAPK with SB203580 blocked flinching and guarding behaviors in a dose-dependent manner whereas no effect on thresholds to tactile stimuli was observed. Radiographic analyses of bones demonstrated that chronic inhibition of p38 MAPK reduced bone loss and incidence of spontaneous fracture in cancer-treated mice. Histological analysis of bones collected from mice treated with the p38 MAPK inhibitor showed complete absence of osteoblastic growth in the intramedullary space as well as significantly reduced tumor burden.
Blockade of non-evoked pain behaviors but not hypersensitivity suggests differences in the underlying mechanisms of specific components of the pain syndrome and a possibility to individualize aspects of pain management. While it is not known whether the role of p38 MAPK signaling can be expanded to other cancers, the data suggest a need for understanding molecular mechanisms and cellular events that initiate and maintain cancer-induced bone pain for effective management for both ongoing pain as well as breakthrough pain.
The unparalleled success of combination antiretroviral therapy (cART) is based on the combination of three drugs from two classes. There is insufficient evidence whether simplification to ritonavir boosted protease inhibitor (PI/r) monotherapy in virologically suppressed HIV-infected patients is effective and safe to reduce cART side effects and costs.
We systematically searched Medline, Embase, the Cochrane Library, conference proceedings and trial registries to identify all randomised controlled trials comparing PI/r monotherapy to cART in suppressed patients. We calculated in an intention to treat (loss-of follow-up, discontinuation of assigned drugs equals failure) and per-protocol analysis (exclusion of protocol violators following randomisation) and based on three different definitions for virological failure pooled risk ratios for remaining virologically suppressed.
We identified 10 trials comparing 3 different PIs with cART based on a PI/r plus 2 reverse transcriptase inhibitors in 1189 patients. With the most conservative approach (viral load <50 copies/ml on two consecutive measurements), the risk ratios for viral suppression at 48 weeks of PI/r monotherapy compared to cART were in the ITT analysis 0.94 8 (95% CI 0.89 to 1.00) p = 0.06; risk difference −0.06 (95%CI -0.11 to 0) p = 0.05, p for heterogeneity = 0.08, I2 = 43.1%) and in the PP analysis 0.93 ((95%CI 0.90 to 0.97) p<0.001; risk difference −0.07 (95%CI −0.10 to −0.03) p<0.001, p for heterogeneity = 0.44, I2 = 0%). Reintroduction of cART in 44 patients with virological failure led in 93% to de-novo viral suppression.
Virologically well suppressed HIV-infected patients have a lower chance to maintain viral suppression when switching from cART to PI/r monotherapy. Failing patients achieve high rates of de-novo viral suppression following reintroduction of reverse transcriptase inhibitors.
Increasing blood pressure has a continuum of adverse risk for cardiovascular events. Traditionally this single measure was used to determine who to treat and how vigorously. However, estimating absolute risk rather than measurement of a single risk factor such as blood pressure is a superior method to identify who is most at risk of having an adverse cardiovascular event such as stroke or myocardial infarction, and therefore who would most likely benefit from therapeutic intervention. Cardiovascular disease (CVD) risk calculators must be used to estimate absolute risk in those without overt CVD as physician estimation is unreliable. Incorporation into usual practice and limitations of the strategy are discussed.
Cannabinoid CB2 agonists have been shown to alleviate behavioral signs of inflammatory and neuropathic pain in animal models. AM1241, a CB2 agonist, does not demonstrate central nervous system side-effects seen with CB1 agonists such as hypothermia and catalepsy. Metastatic bone cancer causes severe pain in patients and is treated with analgesics such as opiates. Recent reports suggest that sustained opiates can produce paradoxical hyperalgesic actions and enhance bone destruction in a murine model of bone cancer. In contrast, CB2 selective agonists have been shown to reduce bone loss associated with a model of osteoporosis. Here we tested whether a CB2 agonist administered over a 7 day period inhibits bone cancer-induced pain as well as attenuates cancer-induced bone degradation.
A murine bone cancer model was used in which osteolytic sarcoma cells were injected into the intramedullary space of the distal end of the femur. Behavioral and radiographic image analysis was performed at days 7, 10 and 14 after injection of tumor cells into the femur.
Osteolytic sarcoma within the femur produced spontaneous and touch evoked behavioral signs of pain within the tumor-bearing limb. The systemic administration of AM1241 acutely or for 7 days significantly attenuated spontaneous and evoked pain in the inoculated limb. Sustained AM1241 significantly reduced bone loss and decreased the incidence of cancer-induced bone fractures.
These findings suggest a novel therapy for cancer-induced bone pain, bone loss and bone fracture while lacking many unwanted side effects seen with current treatments for bone cancer pain.
CB2 agonists; AM1241; osteolytic sarcoma; bone cancer pain
The heterogeneous nuclear ribonucleoprotein (hnRNP) K is an essential RNA and DNA binding protein involved in gene expression and signal transduction including DNA transcription, RNA splicing, RNA stability, and translation. The role of hnRNP K in cancer is relatively understudied. However, several cellular functions strongly indicate that hnRNP K is involved in tumorigenesis. In the present study, we investigated the altered protein expression and the subcellular distribution of the hnRNP K protein using tissue microarrays in pancreatic cancer. We showed an increased cytoplasmic hnRNP K in pancreatic cancer. This increase in hnRNP K protein occurs at the posttranscriptional level. We postulate that the cytoplasmic accumulation of hnRNP K will lead to silenced mRNA translation of tumor suppressor genes and thus contributes to pancreatic cancer development. We also demonstrated that knocking down of hnRNP K expression by siRNA inhibited pancreatic cancer cell growth and colony formation. hnRNP K was identified as a member of the p53/HDM2 pathway. Whether hnRNP K interacts with the mutant p53 is not known. Using two different pancreatic cancer cell lines, we can demonstrate that hnRNP K interacts with the mutant p53. The subcellular distribution and function of the mutant p53 and the interaction of hnRNP K/mutant p53 were affected by the Ras/MEK/ERK pathway, growth factors and the specific p53 mutations in pancreatic cancer cells. Since Kras is activated and p53 is mutated in most pancreatic cancers, these data unveiled an important new signaling pathway that linked by hnRNP K and mutant p53 in pancreatic cancer tumorigenesis.
heterogeneous nuclear ribonucleoprotein K; tumorigenesis; mutant p53; pancreatic cancer
The goal of this study was to develop an in vivo awake mouse model for extracellular bladder sensory nerve recording. A bipolar 125-μm silver electrode was positioned under a single postganglionic bladder nerve. Efferent nerve signals were eliminated by tying off the postganglionic bladder nerve between the major pelvic ganglion and the recording electrode. Sensory nerve activity was measured in the conscious animals 48 h after surgery during continuous intravesical infusion of 0.9% saline/0.5% acetic acid followed by 0.5% acetic acid with capsazepine (10 μM) at a rate of 0.75 ml/h. Continuous infusion of 0.9% NaCl led to a gradual increase in the frequency of sensory nerve firing that peaked upon reaching threshold pressure. Non-micturition contractions were observed in some animals during filling and other animals exhibited only minimal pressure fluctuations; both types of events were associated with a rise in sensory nerve activity. Intravesical infusion of 0.5% acetic acid reduced the intermicturition interval. This was associated with a 2.1-fold increase in bladder pressure during filling and a two-fold increase at both threshold and micturition pressures. Concurrent with these changes, sensory activity increased 2.8-fold during filling and 2.4-fold at threshold pressure. Subsequent intravesical infusion of capsazepine in 0.5% acetic acid reduced filling and threshold pressures by 21 and 31.2%, respectively, and produced corresponding decreases of 36 and 23.4% in sensory nerve activity. The current study shows that multifiber sensory nerve recordings can be reproducibly obtained from conscious mice.
urinary bladder; sensory nerves; conscious mouse
The purpose of this study was to elucidate the mechanisms by which ATP increases guinea pig gallbladder smooth muscle (GBSM) excitability. We evaluated changes in membrane potential and action potential frequency in GBSM using intracellular recording. Application of ATP (100 µM) caused membrane depolarization and a significant increase in AP frequency that were not sensitive to block by tetrodotoxin (0.5 µM). The non-selective P2 antagonist, suramin (100 µM), blocked the excitatory response, resulting in decreased AP frequency in the presence of ATP. The excitatory response to ATP was not altered by PPADS (30 µM), a non-selective P2X antagonist. UTP also caused membrane depolarization and increased AP frequency, with a similar dose-response relationship as ATP. RT-PCR demonstrated that the P2Y4, but not P2Y2, receptor subtype is expressed in guinea pig gallbladder muscularis. ATP induced excitation was blocked by indomethacin (10 µM) and the COX-1 inhibitor, SC-560 (300 nM), but not the COX-2 inhibitor, nimesulide (500 nM). These data suggest that ATP stimulates P2Y4 receptors within the gallbladder muscularis and, in turn, stimulate prostanoid production via COX-1 leading to increased excitability of GBSM.
Esophageal adenocarcinoma risk in Barrett’s esophagus (BE) is increased 30- to 125-fold versus the general population. Among all BE patients, however, neoplastic progression occurs only once per 200 patient-years. Molecular biomarkers are therefore needed to risk-stratify patients for more efficient surveillance endoscopy and to improve the early detection of progression. We therefore performed a retrospective, multicenter, double-blinded validation study of 8 BE progression prediction methylation biomarkers. Progression or nonprogression were determined at 2 years (tier 1) and 4 years (tier 2). Methylation was assayed in 145 nonprogressors (NPs) and 50 progressors (Ps) using real-time quantitative methylation-specific PCR. Ps were significantly older than NPs (70.6 vs. 62.5 years, p < 0.001). We evaluated a linear combination of the 8 markers, using coefficients from a multivariate logistic regression analysis. Areas under the ROC curve (AUCs) were high in the 2-, 4-year and combined data models (0.843, 0.829 and 0.840; p<0.001, p<0.001 and p<0.001, respectively). In addition, even after rigorous overfitting correction, the incremental AUCs contributed by panels based on the 8 markers plus age vs. age alone were substantial (Δ-AUC = 0.152, 0.114 and 0.118, respectively) in all three models. A methylation biomarker-based panel to predict neoplastic progression in BE has potential clinical value in improving both the efficiency of surveillance endoscopy and the early detection of neoplasia.