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1.  Incomplete Reversibility of Estimated Glomerular Filtration Rate Decline Following Tenofovir Disoproxil Fumarate Exposure 
Jose, Sophie | Hamzah, Lisa | Campbell, Lucy J. | Hill, Teresa | Fisher, Martin | Leen, Clifford | Gilson, Richard | Walsh, John | Nelson, Mark | Hay, Phillip | Johnson, Margaret | Chadwick, David | Nitsch, Dorothea | Jones, Rachael | Sabin, Caroline A. | Post, Frank A. | Ainsworth, Jonathan | Anderson, Jane | Babiker, Abdel | Chadwick, David | Delpech, Valerie | Dunn, David | Fisher, Martin | Gazzard, Brian | Gilson, Richard | Gompels, Mark | Hay, Phillip | Hill, Teresa | Johnson, Margaret | Kegg, Stephen | Leen, Clifford | Nelson, Mark | Orkin, Chloe | Palfreeman, Adrian | Phillips, Andrew | Pillay, Deenan | Post, Frank | Sabin, Caroline | Sachikonye, Memory | Schwenk, Achim | Walsh, John | Hill, Teresa | Huntington, Susie | Josie, Sophie | Phillips, Andrew | Sabin, Caroline | Thornton, Alicia | Dunn, David | Glabay, Adam | Orkin, C. | Garrett, N. | Lynch, J. | Hand, J. | de Souza, C. | Fisher, M. | Perry, N. | Tilbury, S. | Churchill, D. | Gazzard, B. | Nelson, M. | Waxman, M. | Asboe, D. | Mandalia, S. | Delpech, V. | Anderson, J. | Munshi, S. | Korat, H. | Poulton, M. | Taylor, C. | Gleisner, Z. | Campbell, L. | Babiker, Abdel | Dunn, David | Glabay, Adam | Gilson, R. | Brima, N. | Williams, I. | Schwenk, A. | Ainsworth, J. | Wood, C. | Miller, S. | Johnson, M. | Youle, M. | Lampe, F. | Smith, C. | Grabowska, H. | Chaloner, C. | Puradiredja, D. | Walsh, J. | Weber, J. | Ramzan, F. | Mackie, N. | Winston, A. | Leen, C. | Wilson, A. | Gompels, M. | Allan, S. | Palfreeman, A. | Moore, A. | Chadwick, D. | Wakeman, K. | Kegg, Stephen | Main, Paul | Mitchell,  | Hunter,  | Sachikonye, Memory | Hay, Phillip | Dhillon, Mandip
The Journal of Infectious Diseases  2014;210(3):363-373.
Background. Tenofovir disoproxil fumarate (TDF) has been linked to renal impairment, but the extent to which this impairment is reversible is unclear. We aimed to investigate the reversibility of renal decline during TDF therapy.
Methods. Cox proportional hazards models assessed factors associated with discontinuing TDF in those with an exposure duration of >6 months. In those who discontinued TDF therapy, linear piecewise regression models estimated glomerular filtration rate (eGFR) slopes before initiation of, during, and after discontinuation of TDF therapy. Factors associated with not achieving eGFR recovery 6 months after discontinuing TDF were assessed using multivariable logistic regression.
Results. We observed declines in the eGFR during TDF exposure (mean slopes, −15.7 mL/minute/1.73 m2/year [95% confidence interval {CI}, −20.5 to −10.9] during the first 3 months and −3.1 mL/minute/1.73 m2/year [95% CI, −4.6 to −1.7] thereafter) and evidence of eGFR increases following discontinuation of TDF therapy (mean slopes, 12.5 mL/minute/1.73 m2/year [95% CI, 8.9–16.1] during the first 3 months and 0.8 mL/minute/1.73 m2/year [95% CI, .1–1.5] thereafter). Following TDF discontinuation, 38.6% of patients with a decline in the eGFR did not experience recovery. A higher eGFR at baseline, a lower eGFR after discontinuation of TDF therapy, and more-prolonged exposure to TDF were associated with an increased risk of incomplete recovery 6 months after discontinuation of TDF therapy.
Conclusions. This study shows that a decline in the eGFR during TDF therapy was not fully reversible in one third of patients and suggests that prolonged TDF exposure at a low eGFR should be avoided.
PMCID: PMC4091582  PMID: 24585896
tenofovir; highly active antiretroviral therapy; eGFR; eGFR slopes; renal function; kidney
2.  5-year Safety Evaluation of Maraviroc in HIV-1-Infected, Treatment-Experienced Patients 
Maraviroc is unique among approved antiretroviral drugs in targeting the host-cell CCR5 receptor. With its novel mechanism of action, we sought to describe the 5-year safety profile of maraviroc.
Two large phase 3 studies of maraviroc enrolled HIV-infected, treatment-experienced patients and followed them for 5 or more years. Survival and selected clinical endpoints were identified and assessed.
A total of 938 enrolled patients received maraviroc-containing regimens. Rates of death and selected clinical events (e.g., hepatic failure, malignancy, myocardial infarction) were low during follow-up.
Maraviroc was generally safe in treatment-experienced participants over more than 5 years.
PMCID: PMC3893710  PMID: 24419064
maraviroc; CCR5 antagonist; HIV; antiretroviral therapy; safety; treatment-experienced patients
3.  A PLCγ1-Dependent, Force-Sensitive Signaling Network in the Myogenic Constriction of Cerebral Arteries 
Science signaling  2014;7(327):ra49.
Maintaining constant blood flow in the face of fluctuations in blood pressure is a critical autoregulatory feature of cerebral arteries. An increase in pressure within the artery lumen causes the vessel to constrict through depolarization and contraction of the encircling smooth muscle cells. This pressure-sensing mechanism involves activation of two types of transient receptor potential (TRP) channels: TRPC6 and TRPM4. We provide evidence that the activation of the γ1 isoform of phospholipase C (PLCγ1) is critical for pressure sensing in cerebral arteries. Inositol 1,4,5-trisphosphate (IP3), generated by PLCγ1 in response to pressure, sensitized IP3 receptors (IP3Rs) to Ca2+ influx mediated by the mechanosensitive TRPC6 channel, synergistically increasing IP3R-mediated Ca2+ release to activate TRPM4 currents, leading to smooth muscle depolarization and constriction of isolated cerebral arteries. Proximity ligation assays demonstrated colocalization of PLCγ1 and TRPC6 with TRPM4, suggesting the presence of a force-sensitive, local signaling network comprising PLCγ1, TRPC6, TRPM4, and IP3Rs. Src tyrosine kinase activity was necessary for stretch-induced TRPM4 activation and myogenic constriction, consistent with the ability of Src to activate PLCγ isoforms. We conclude that contraction of cerebral artery smooth muscle cells requires the integration of pressure-sensing signaling pathways and their convergence on IP3Rs, which mediate localized Ca2+-dependent depolarization through the activation of TRPM4.
PMCID: PMC4170587  PMID: 24866019
4.  Preferential, enhanced breast cancer cell migration on biomimetic electrospun nanofiber ‘cell highways’ 
BMC Cancer  2014;14(1):825.
Aggressive metastatic breast cancer cells seemingly evade surgical resection and current therapies, leading to colonization in distant organs and tissues and poor patient prognosis. Therefore, high-throughput in vitro tools allowing rapid, accurate, and novel anti-metastatic drug screening are grossly overdue. Conversely, aligned nanofiber constitutes a prominent component of the late-stage breast tumor margin extracellular matrix. This parallel suggests that the use of a synthetic ECM in the form of a nanoscale model could provide a convenient means of testing the migration potentials of cancer cells to achieve a long-term goal of providing clinicians an in vitro platform technology to test the efficacy of novel experimental anti-metastatic compounds.
Electrospinning produces highly aligned, cell-adhesive nanofiber matrices by applying a strong electric field to a polymer-containing solution. The resulting fibrous microstructure and morphology closely resembles in vivo tumor microenvironments suggesting their use in analysis of migratory potentials of metastatic cancer cells. Additionally, a novel interface with a gel-based delivery system creates CXCL12 chemotactic gradients to enhance CXCR4-expressing cell migration.
Cellular dispersions of MCF-10A normal mammary epithelial cells or human breast cancer cells (MCF-7 and MDA-MB-231) seeded on randomly-oriented nanofiber exhibited no significant differences in total or net distance traveled as a result of the underlying topography. Cells traveled ~2-5 fold greater distances on aligned fiber. Highly-sensitive MDA-MB-231 cells displayed an 82% increase in net distance traversed in the presence of a CXCL12 gradient. In contrast, MCF-7 cells exhibited only 31% increase and MCF-10A cells showed no statistical difference versus control or vehicle conditions. MCF-10A cells displayed little sensitivity to CXCL12 gradients, while MCF-7 cells displayed early sensitivity when CXCL12 concentrations were higher. MDA-MB-231 cells displayed low relative expression levels of CXCR4, but high sensitivity resulting in 55-fold increase at late time points due to CXCL12 gradient dissipation.
This model could create clinical impact as an in vitro diagnostic tool for rapid assessment of tumor needle biopsies to confirm metastatic tumors, their invasiveness, and allow high-throughput drug screening providing rapid development of personalized therapies.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-825) contains supplementary material, which is available to authorized users.
PMCID: PMC4236463  PMID: 25385001
Topography; Cell motility; Chemotaxis; MDA-MB-231; MCF-10A; MCF-7
5.  Impact of NRTI backbone on renal, bone and cardiovascular markers in HIV-infected individuals receiving a boosted protease inhibitor 
Journal of the International AIDS Society  2014;17(4Suppl 3):19562.
We have previously shown in the SSAT 044 study that unconjugated hyperbilirubinaemia in subjects receiving a boosted protease inhibitor (PI/r) has limited impact on renal, cardiovascular (CV) and bone biomarkers, as well as on neurocognitive performance, relative to those receiving PI/r with a normal bilirubin. We present here a secondary analysis comparing markers in those receiving abacavir- vs tenofovir- based antiretroviral therapy (ART).
Materials and Methods
This cross-sectional study included 101 HIV-1 infected individuals stable (HIV RNA<50 cps/ml, >6 months) on antiretroviral regimens including tenofovir (TDF)/emtricitabine or abacavir/lamivudine plus a ritonavir boosted PI.
Forty-three subjects had normal bilirubin (NBR) levels and 35 had high bilirubin (>2.5 times upper limit); the remaining 23 patients had intermediate bilirubin levels or violated the protocol. The mean age of participants was 48 years; 93% were male and 84% Caucasian; 22 received ABC-based therapy and 78 TDF. No differences were seen in cardiovascular markers: Framingham (10-year risk % median, IQR): ABC 8.1, 5.6–15.3; TDF 9.5, 4.8–13.4 (p=ns); pulse wave velocity and carotid intimal thickness also showed no significant differences. No differences were seen in bone parameters: Calcaneal Stiffness Index (median score, IQR): ABC −0.5, −0.8 to 0.8; TDF −0.5, 1.4–0.4 (p=ns); 10 year FRAX score (% median, IQR): ABC 5.0, 2.4–6.2; TDF 3.6, 2.5–5.8 (p=ns). There were differences in renal parameters as shown in Table 1. We show statistically significant differences in urine protein/creatinine ratio (uPCR) (10 vs 7; p=0.004) and urine albumin/creatinine ratio (uACR) (15 vs 8; p=0.002), with both being higher in the TDF group.
Tenofovir use is associated with excess loss of proteins including those typically resorbed in the renal tubule. Abacavir use was not associated with an increase in biomarkers of CV risk or vascular dysfunction.
PMCID: PMC4224792  PMID: 25394069
6.  Telaprevir combination therapy in HCV/HIV co-infected patients (INSIGHT study): sustained virologic response at 12 weeks final analysis 
Journal of the International AIDS Society  2014;17(4Suppl 3):19626.
We report the SVR12 final analysis of a phase 3 study of telaprevir in combination with peginterferon (P)/ribavirin (R) in HCV-genotype 1, treatment-naïve and -experienced patients with HCV/HIV co-infection (INSIGHT).
Materials and Methods
Patients receiving stable, suppressive HIV antiretroviral (ARV) therapy, containing atazanavir/ritonavir, efavirenz, darunavir/ritonavir, raltegravir, etravirine or rilpivirine, received telaprevir 750 mg q8h (1125 mg q8h if on efavirenz) plus P (180 µg once-weekly) and R (800 mg/day) for 12 weeks, followed by an additional 12 weeks (non-cirrhotic HCV treatment-naïve and relapse patients with extended rapid viral response [eRVR]) or 36 weeks (all others) of PR alone. Analysis was performed when all patients had completed the follow-up visit of 12 weeks after last planned dose.
One hundred sixty-two patients were enrolled and treated (65 efavirenz, 59 atazanavir/ritonavir, 17 darunavir/ritonavir, 17 raltegravir, 4 etravirine). Mean age was 45 years, 78% were male, 92% were Caucasian; mean CD4 count was 687 cells/mm3. Sixty four patients (40%) were HCV treatment-naïve and 98 (60%) were treatment experienced (29 relapsers, 18 partial responders and 51 null responders). 64% were subtype 1a. 30% had bridging fibrosis (17%) or cirrhosis (13%). 19% of patients discontinued telaprevir, including 9% due to an adverse event (AE), 8% reaching a virologic endpoint and 2% for other reasons (non compliance or not defined). Treatment responses are shown in Table 1. There were no HIV RNA breakthroughs. Most frequently reported (≥20% patients) AEs were pruritus 43%; fatigue 27%; rash 34%, anorectal events 30% and influenza-like illness (25%). Anemia was reported in 15% of patients; grade ≥3 haemoglobin decrease occurred in 2.5% of patients. 6% of patients experienced serious AEs.
In this phase 3 study of HIV-infected, HCV treatment-naïve and -experienced patients, 49% achieved eRVR and 57% reached SVR12. In patients with an eRVR, SVR12 rates were >80%, irrespective of prior treatment history.
PMCID: PMC4224845  PMID: 25394130
7.  A comparison of inpatient admissions in 2012 from two European countries 
Journal of the International AIDS Society  2014;17(4Suppl 3):19712.
This study compares the trends of HIV inpatient admissions between a London tertiary HIV centre (United Kingdom) and four infectious disease wards in Italy (IT) to recognize common patterns across Europe.
Data regarding HIV inpatient admissions was collected by using discharge diagnostic codes from 1 January to 31 December 2012, including patient demographics, combined antiretroviral therapy (cART) history, CD4, viral load (VL) and mortality rates. Discharge diagnoses were categorized according to the International Classification of Disease (ICD) 9 and 10 system. All ICD categories that reach a 3% threshold of total admissions were analyzed.
A total of 731 admissions (257 in Italy and 474 in the United Kingdom) for 521 patients (1.5 mean admission per patient). Female admissions were higher in Italy at 22.6% (n=58) compared to 14.9% (n=47) in the United Kingdom. Median age of patients was 47 years old. There was an undetectable VL in 65.8% (n=169) of admissions in Italy and 67.1% (n=319) in the United Kingdom (p=0.385); 86.4% (n=222) and 82.4% (n=389) of admissions were on cART, respectively. Mean CD4 was 302 in Italy compared to 368 in the United Kingdom (p=0.003). Average length of admission was 16 days with a 10.2% (n=21) mortality rate in Italy compared to 8 days with 2.8% (n=9) mortality in the United Kingdom (p<0.001). HCV co-infection was present in 64.6% (n=166) in Italy and 13.5% (n=64) in the United Kingdom and commonest mode of transmission was needle use in Italy (67.3%, n=173) and men who have sex with men in the UK cohort (59.9%, n=284). The cause of inpatient admissions according to ICD codes can be seen in following Figure 1.
Significant differences in the duration of inpatient admission and mortality rates can be observed between these two cohorts which is secondary to the impact of Hepatitis C co-infection in Italy. However increases in the number of Hepatitis C co-infection patients amongst MSM in London has been reported [1] and route of transmission in Italy is shifting towards MSM [2], therefore it is important to learn how HIV is developing and managed in a global context to help plan future for services. The UK cohort demonstrates a wider range of conditions necessitating admission, and with an ageing HIV population, this is expected to increase in the future, requiring general and specialist HIV physicians to work closely together. The HIV-RNA threshold is 400 copies/mL to account for blips according to British HIV Association (BHIVA) Guidelines 2012 [3].
PMCID: PMC4225254  PMID: 25397459
8.  Detection of resistance mutations and CD4 slopes in individuals experiencing sustained virological failure 
Journal of the International AIDS Society  2014;17(4Suppl 3):19737.
Several resistance mutations have been shown to affect viral fitness, and the presence of certain mutations might result in clinical benefit for patients kept on a virologically failing regimen due to an exhaustion of drug options. We sought to quantify the effect of resistance mutations on CD4 slopes in patients undergoing episodes of viral failure.
Materials and Methods
Patients from the EuroSIDA and UK CHIC cohorts undergoing at least one episode of virological failure (>3 consecutive RNA measurements >500 on ART) with at least three CD4 measurements and a resistance test during the episode were included. Mutations were identified using the IAS-US (2013) list, and were presumed to be present from detection until the end of an episode. Multivariable linear mixed models with a random intercept and slope adjusted for age, baseline CD4 count, hepatitis C, drug type, RNA (log-scale), risk group and subtype were used to estimate CD4 slopes. Individual mutations with a population prevalence of >10% were tested for their effect on the CD4 slope.
A total of 2731 patients experiencing a median of 1 (range 1–4) episodes were included in this analysis. The prevalence of any resistance per episode was 88.4%; NNRTI resistance was most common (78.5%). Overall, CD4 counts declined by 17.1 (−19.7; −14.5) cells per year; this decline was less marked with partial viral suppression (current HIV RNA more than 1.5 log below the setpoint; p=0.01). In multivariable models adjusting for viral load, CD4 decline was slower during episodes with detected resistance compared to episodes without detected resistance (21.0 cells/year less, 95% CI 11.75–30.31, p<0.001). Among those with more than one resistance mutation, there was only weak evidence that class-specific mutations had any effect on the CD4 slope (Table 1). The effects of individual mutations (incl. M184V) were explored, but none were significantly associated with the CD4 slope; for these comparisons, a Bonferroni-corrected p-value level was 0.003.
In our study population, detected resistance was associated with slightly less steep CD4 declines. This may be due to a biological effect of resistance on CD4 slopes, or other unmeasured factors such as poor adherence among individuals without resistance. Among individuals with detected drug resistance, we found no evidence suggesting that the presence of individual mutations was associated with beneficial CD4 slope changes.
PMCID: PMC4225350  PMID: 25397482
9.  Effect of hyperbilirubinaemia on neurocognitive, renal, bone and cardiovascular markers in HIV infection treated with boosted protease inhibitors 
Journal of the International AIDS Society  2014;17(4Suppl 3):19827.
Use of some protease inhibitors (PI) is associated with unconjugated hyperbilirubinaemia (HBR), due to inhibition of UGT1A1. As observed in Gilbert's syndrome, HBR may have antioxidant and anti-inflammatory effects. Inflammation may be relevant to neurocognitive (NC) impairment, cardiovascular, renal and bone co-morbidities in HIV infection. This study aimed to analyse correlations between antiretroviral associated HBR and NC impairment as well as renal, bone and cardiovascular parameters.
Material and Methods
This cross-sectional study included 101 HIV-1-infected individuals stable (>6 months) on antiretroviral regimens including tenofovir/emtricitabine or abacavir/lamivudine plus a ritonavir-boosted PI. Patients with >grade 2 HBR were compared to patients with normal bilirubin on NC data collected using CogState. An overall composite score was calculated for each subject. Two-tail P-values were calculated using the Mann-Whitney U test. We measured the following parameters in all participants: Bone – Calcaneal Stiffness Index (CSI), blood bone markers, calculated FRAX score; CV – vascular endothelial function markers (iCAM, vCAM), lipid fractions and sub fractions (Total, HDL and LDL cholesterol, triglycerides, ApoB), Carotid Intimal Thickness (CIT), Pulse Wave Velocity (PWV), glucose and insulin for calculation of HOMA-IR, IL-6, d-dimer, uric acid, and hsCRP; Renal – urea and electrolytes (U&E), urinary protein/creatinine ratio (uPCR), urinary retinal binding protein (RBP)/creatinine ratio.
Forty-three participants had normal bilirubin (NBR) levels and 35 had high bilirubin (HBR; >2.5 times upper limit); the remaining 23 patients had intermediate bilirubin levels or violated the protocol. The mean age of participants was 48 years; 93% were male and 84% Caucasian. Mostly no significant differences were seen in any of the markers when comparing the NBR and HBR groups. Two component tests of the CogState were seen to be different – visual learning and memory (OCL) and working memory (ONB) (Table 1).
VCAM1 and FRAX scores were noted to be different between the two groups, but the latter had a worse outcome in the HBR group.
The numbers seen here were not felt to be large enough to draw clear conclusions around clinical significance. In the context of overall cognitive screening, the individual test differences were also not felt to be clinically significant. Clearly there are some early signs here of differences that may be worth investigating further in a larger, prospective study.
PMCID: PMC4225352  PMID: 25397571
10.  Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle 
The Journal of General Physiology  2014;143(5):559-575.
Angiotensin signaling promotes interactions between AKAP150, PKC, and TRPV4 channels to form signaling domains that control Ca2+ influx into arterial myocytes.
Transient receptor potential vanilloid 4 (TRPV4) channels are Ca2+-permeable, nonselective cation channels expressed in multiple tissues, including smooth muscle. Although TRPV4 channels play a key role in regulating vascular tone, the mechanisms controlling Ca2+ influx through these channels in arterial myocytes are poorly understood. Here, we tested the hypothesis that in arterial myocytes the anchoring protein AKAP150 and protein kinase C (PKC) play a critical role in the regulation of TRPV4 channels during angiotensin II (AngII) signaling. Super-resolution imaging revealed that TRPV4 channels are gathered into puncta of variable sizes along the sarcolemma of arterial myocytes. Recordings of Ca2+ entry via single TRPV4 channels (“TRPV4 sparklets”) suggested that basal TRPV4 sparklet activity was low. However, Ca2+ entry during elementary TRPV4 sparklets was ∼100-fold greater than that during L-type CaV1.2 channel sparklets. Application of the TRPV4 channel agonist GSK1016790A or the vasoconstrictor AngII increased the activity of TRPV4 sparklets in specific regions of the cells. PKC and AKAP150 were required for AngII-induced increases in TRPV4 sparklet activity. AKAP150 and TRPV4 channel interactions were dynamic; activation of AngII signaling increased the proximity of AKAP150 and TRPV4 puncta in arterial myocytes. Furthermore, local stimulation of diacylglycerol and PKC signaling by laser activation of a light-sensitive Gq-coupled receptor (opto-α1AR) resulted in TRPV4-mediated Ca2+ influx. We propose that AKAP150, PKC, and TRPV4 channels form dynamic subcellular signaling domains that control Ca2+ influx into arterial myocytes.
PMCID: PMC4003184  PMID: 24778429
Members of the transient receptor potential (TRP) channel superfamily are present in vascular smooth muscle cells and play important roles in the regulation of vascular contractility.The TRPC3 and TRPC6 channels are activated by stimulation of several excitatory receptors in vascular smooth muscle cells. Activation of these channels leads to myocyte depolarization, which stimulates Ca2+ entry via voltage-dependent Ca2+ channels (VDCC), leading to vasoconstriction. The TRPV4 channels in arterial myocytes are activated by epoxyeicosatrienoic acids, and activation of the channels enhances Ca2+ spark and transient Ca2+-sensitive K+ channel activity, thereby hyperpolarizing and relaxing vascular smooth muscle cells.The TRPC6 and TRPM4 channels are activated by mechanical stimulation of cerebral artery myocytes. Subsequent depolarization and activation of VDCC Ca2+ entry is directly linked to the development of myogenic tone in vitro and to autoregulation of cerebral blood flow in vivo.These findings imply a fundamental importance of TRP channels in the regulation of vascular smooth muscle tone and suggest that TRP channels could be important targets for drug therapy under conditions in which vascular contractility is disturbed (e.g. hypertension, stroke, vasospasm).
PMCID: PMC4193799  PMID: 18215190
autoregulation; cation channels; myogenic tone; transient receptor potential (TRP) channels; vascular Ca2+ regulation
12.  Mechanisms of enhanced basal tone of brain parenchymal arterioles during early postischemic reperfusion: role of ET-1-induced peroxynitrite generation 
The contributions of vasoconstrictors (endothelin-1 (ET-1), peroxynitrite) and endothelium-dependent vasodilatory mechanisms to basal tone were investigated in parenchymal arterioles (PAs) after early postischemic reperfusion. Transient middle cerebral artery occlusion (tMCAO) was induced for 2 hours with 30 minutes reperfusion in male Wistar rats and compared with ischemia alone (permanent MCAO (pMCAO); 2.5 hours) or sham controls. Changes in lumen diameter of isolated and pressurized PAs were compared. Quantitative PCR was used to measure endothelin type B (ETB) receptors. Constriction to intravascular pressure (‘basal tone') was not affected by tMCAO or pMCAO. However, constriction to inhibitors of endothelial cell, small- (SK) and intermediate- (IK) conductance, Ca2+-sensitive K+ channels (apamin and TRAM-34, respectively) were significantly enhanced in PAs from tMCAO compared with pMCAO or sham. Addition of the ETB agonist sarafotoxin caused constriction in PAs from tMCAO but not from sham animals (21±4% versus 3±3% at 1 nmol/L; P<0.01) that was inhibited by the peroxynitrite scavenger FeTMPyP (5,10,15,20-tetrakis (N-methyl-4′-pyridyl) porphinato iron (III) chloride) (100 μmol/L). Expression of ETB receptors was not found on PA smooth muscle, suggesting that constriction to sarafotoxin after tMCAO was due to peroxynitrite and not ETB receptor expression. The maintenance of basal tone in PAs after tMCAO may restrict flow to the ischemic region and contribute to infarct expansion.
PMCID: PMC3790940  PMID: 23778163
EDH; endothelin-1; focal ischemia; parenchymal arterioles; peroxynitrite
13.  How MicroRNAs Influence both Hereditary and Inflammatory-mediated Colon Cancer 
Cancer genetics  2013;206(0):309-316.
MicroRNAs have emerged as important post-translational regulators of gene expression and are involved in several physiological and pathological states including the pathogenesis of human colon cancers. In regards to tumor development, microRNAs can act as oncogenes or tumor suppressors. Both hereditary predispositions (i.e. Lynch Syndrome and Familial Adenomatous Polyposis) contribute to the development of colon cancer. In addition, individuals who suffer from inflammatory bowel diseases such as Crohn’s disease or ulcerative colitis have a higher risk of developing colon cancer. Here, we discuss the occurrence of the deregulated expression of miRNAs in colon cancer that arise due to hereditary predisposition and inflammatory bowel disease.
PMCID: PMC3893936  PMID: 24042167
MicroRNA; HNPCC; FAP; Crohn’s Disease; Ulcerative Colitis
14.  Mimicking white matter tract topography using core–shell electrospun nanofibers to examine migration of malignant brain tumors 
Biomaterials  2013;34(21):5181-5190.
Glioblastoma multiforme (GBM), one of the deadliest forms of human cancer, is characterized by its high infiltration capacity, partially regulated by the neural extracellular matrix (ECM). A major limitation in developing effective treatments is the lack of in vitro models that mimic features of GBM migration highways. Ideally, these models would permit tunable control of mechanics and chemistry to allow the unique role of each of these components to be examined. To address this need, we developed aligned nanofiber biomaterials via core–shell electrospinning that permit systematic study of mechanical and chemical influences on cell adhesion and migration. These models mimic the topography of white matter tracts, a major GBM migration ‘highway’. To independently investigate the influence of chemistry and mechanics on GBM behaviors, nanofiber mechanics were modulated by using different polymers (i.e., gelatin, poly(ethersulfone), poly(dimethylsiloxane)) in the ‘core’ while employing a common poly(ε-caprolactone) (PCL) ‘shell’ to conserve surface chemistry. These materials revealed GBM sensitivity to nanofiber mechanics, with single cell morphology (Feret diameter), migration speed, focal adhesion kinase (FAK) and myosin light chain 2 (MLC2) expression all showing a strong dependence on nanofiber modulus. Similarly, modulating nanofiber chemistry using extracellular matrix molecules (i.e., hyaluronic acid (HA), collagen, and Matrigel) in the ‘shell’ material with a common PCL ‘core’ to conserve mechanical properties revealed GBM sensitivity to HA; specifically, a negative effect on migration. This system, which mimics the topographical features of white matter tracts, should allow further examination of the complex interplay of mechanics, chemistry, and topography in regulating brain tumor behaviors.
PMCID: PMC4080638  PMID: 23601662
Glioblastoma multiforme; White matter; Nanofiber; Mechanics; Chemistry
15.  First Line Therapy in 2014 
BMC Infectious Diseases  2014;14(Suppl 2):S3.
PMCID: PMC4221072
16.  Ryanodine receptors, calcium signaling and regulation of vascular tone in the cerebral parenchymal microcirculation 
The cerebral blood supply is delivered by a surface network of pial arteries and arterioles from which arise (parenchymal) arterioles that penetrate into the cortex and terminate in a rich capillary bed. The critical regulation of cerebral blood flow, locally and globally, requires precise vasomotor regulation of the intracerebral microvasculature. This vascular region is anatomically unique as illustrated by the presence of astrocytic processes that envelope almost the entire basolateral surface of parenchymal arterioles. There are, moreover, notable functional differences between pial arteries and parenchymal arterioles. For example, in pial vascular smooth muscle cells (VSMCs), local calcium release events (“calcium sparks”) through ryanodine receptor (RyR) channels in sarcoplasmic reticulum membrane activate large conductance, calcium-sensitive potassium (BK) channels to modulate vascular diameter. In contrast, VSMCs in parenchymal arterioles express functional RyR and BK channels, but under physiological conditions these channels do not oppose pressure-induced vasoconstriction. Here we summarize the roles of ryanodine receptors in the parenchymal microvasculature under physiologic and pathologic conditions, and discuss their importance in the control of cerebral blood flow.
PMCID: PMC3612564  PMID: 23216877
brain parenchymal arteriole; cerebral blood flow; acidosis; ryanodine receptor; potassium channel
17.  Impact on life expectancy of HIV-1 positive individuals of CD4+ cell count and viral load response to antiretroviral therapy 
AIDS (London, England)  2014;28(8):1193-1202.
The objective of this study is to estimate life expectancies of HIV-positive patients conditional on response to antiretroviral therapy (ART).
Patients aged more than 20 years who started ART during 2000–2010 (excluding IDU) in HIV clinics contributing to the UK CHIC Study were followed for mortality until 2012. We determined the latest CD4+ cell count and viral load before ART and in each of years 1–5 of ART. For each duration of ART, life tables based on estimated mortality rates by sex, age, latest CD4+ cell count and viral suppression (HIV-1 RNA <400 copies/ml), were used to estimate expected age at death for ages 20–85 years.
Of 21 388 patients who started ART, 961 (4.5%) died during 110 697 person-years. At start of ART, expected age at death [95% confidence interval (CI)] of 35-year-old men with CD4+ cell count less than 200, 200–349, at least 350 cells/μl was 71 (68–73), 78 (74–82) and 77 (72–81) years, respectively, compared with 78 years for men in the general UK population. Thirty-five-year-old men who increased their CD4+ cell count in the first year of ART from less than 200 to 200–349 or at least 350 cells/μl and achieved viral suppression gained 7 and 10 years, respectively. After 5 years on ART, expected age at death of 35-year-old men varied from 54 (48–61) (CD4+ cell count <200 cells/μl and no viral suppression) to 80 (76–83) years (CD4+ cell count ≥350 cells/μl and viral suppression).
Successfully treated HIV-positive individuals have a normal life expectancy. Patients who started ART with a low CD4+ cell count significantly improve their life expectancy if they have a good CD4+ cell count response and undetectable viral load.
PMCID: PMC4004637  PMID: 24556869
antiretroviral therapy; CD4+ cell count; HIV; HIV-1 RNA; life expectancy; viral load
18.  Prostaglandin E2, a postulated astrocyte-derived neurovascular coupling agent, constricts rather than dilates parenchymal arterioles 
It has been proposed that prostaglandin E2 (PGE2) is released from astrocytic endfeet to dilate parenchymal arterioles through activation of prostanoid (EP4) receptors during neurovascular coupling. However, the direct effects of PGE2 on isolated parenchymal arterioles have not been tested. Here, we examined the effects of PGE2 on the diameter of isolated pressurized parenchymal arterioles from rat and mouse brain. Contrary to the prevailing assumption, we found that PGE2 (0.1, 1, and 5 μmol/L) constricted rather than dilated parenchymal arterioles. Vasoconstriction to PGE2 was prevented by inhibitors of EP1 receptors. These results strongly argue against a direct role of PGE2 on arterioles during neurovascular coupling.
PMCID: PMC3618402  PMID: 23385200
astrocytes; brain parenchymal arterioles; neurovascular coupling; prostaglandin E2
19.  Etravirine as a Switching Option for Patients with HIV RNA Suppression: A Review of Recent Trials 
AIDS Research and Treatment  2014;2014:636584.
Unlike other nonnucleoside reverse transcriptase inhibitors, etravirine is only approved for use in treatment-experienced patients. In the DUET 1 and 2 trials, 1203 highly treatment-experienced patients were randomized to etravirine or placebo, in combination with darunavir/ritonavir and optimized background treatment. In these trials, etravirine showed significantly higher rates of HIV RNA suppression when compared with placebo (61% versus 40% at Week 48). There was no significant rise of lipids or neuropsychiatric adverse events, but there was an increase in the risk of rash with etravirine treatment. In the SENSE trial, which evaluated etravirine and efavirenz in 157 treatment-naïve patients in combination with 2 nucleoside analogues, there was a lower risk of lipid elevations and neuropsychiatric adverse events with etravirine when compared to efavirenz. Etravirine has been evaluated in three randomized switching studies. In the SSAT029 switch trial, 38 patients who had neuropsychiatric adverse events possibly related to efavirenz showed an improvement in these after switching to etravirine. The Swiss Switch-EE recruited 58 individuals without neuropsychiatric adverse events who were receiving efavirenz, and no benefit was shown when switching to etravirine. In the Spanish ETRA-SWITCH trial (n = 46), there were improvements in lipids when individuals switched from a protease inhibitor to etravirine. These switching trials were conducted in patients with full HIV RNA suppression: <50 copies/mL and with no history of virological failure or resistance to therapy. The results from these three randomized switching studies suggest a possible new role for etravirine, in combination with two nucleoside analogues, as a switching option for those with HIV RNA suppression but who are reporting adverse events possibly related to antiretroviral therapy. However a large well-powered trial would need to be conducted to strengthen the evidence from the pilot studies conducted so far.
PMCID: PMC3955667  PMID: 24715982
20.  Regulation of cerebral cortex size and folding by expansion of basal progenitors 
The EMBO journal  2013;32(13):1817-1828.
Size and folding of the cerebral cortex increased massively during mammalian evolution leading to the current diversity of brain morphologies. Various subtypes of neural stem and progenitor cells have been proposed to contribute differently in regulating thickness or folding of the cerebral cortex during development, but their specific roles have not been demonstrated. We report that the controlled expansion of unipotent basal progenitors in mouse embryos led to megalencephaly, with increased surface area of the cerebral cortex, but not to cortical folding. In contrast, expansion of multipotent basal progenitors in the naturally gyrencephalic ferret was sufficient to drive the formation of additional folds and fissures. In both models, changes occurred while preserving a structurally normal, six-layered cortex. Our results are the first experimental demonstration of specific and distinct roles for basal progenitor subtypes in regulating cerebral cortex size and folding during development underlying the superior intellectual capability acquired by higher mammals during evolution.
PMCID: PMC3926188  PMID: 23624932
basal progenitors; brain evolution; mammalian neural stem cells; mouse brain development; neurogenesis
21.  PyPop: A Software Framework for Population Genomics: Analyzing Large-Scale Multi-Locus Genotype Data 
Software to analyze multi-locus genotype data for entire populations is useful for estimating haplotype frequencies, deviation from Hardy-Weinberg equilibrium and patterns of linkage disequilibrium. These statistical results are important to both those interested in human genome variation and disease predisposition as well as evolutionary genetics. As part of the 13th International Histocompatibility and Immunogenetics Working Group (IHWG), we have developed a software frame-work (PyPop). The primary novelty of this package is that it allows integration of statistics across large numbers of data-sets by heavily utilizing the XML file format and the R statistical package to view graphical output, while retaining the ability to inter-operate with existing software. Largely developed to address human population data, it can, however, be used for population based data for any organism. We tested our software on the data from the 13th IHWG which involved data sets from at least 50 laboratories each of up to 1000 individuals with 9 MHC loci (both class I and class II) and found that it scales to large numbers of data sets well.
PMCID: PMC3891851  PMID: 12603054
22.  Subarachnoid blood converts neurally evoked vasodilation to vasoconstriction in rat brain cortex 
The matching of blood flow to regional brain function, called functional hyperemia or neurovascular coupling, involves the coordinated activity of neurons, astrocytes and parenchymal arterioles. Under physiological conditions, localized neuronal activation leads to elevated astrocyte endfoot Ca2+ and vasodilation, resulting in an increase in cerebral blood flow. In this study, we examined the impact of subarachnoid hemorrhage (SAH) on neurovascular coupling. SAH model rats received two injections of autologous blood into the cisterna magna 24 hours apart. Cortical brain slices from SAH model animals were prepared four days after the initial blood injection. Arteriolar diameter and astrocyte endfoot Ca2+ were simultaneously measured using two-photon microscopy. As expected, neuronal activity evoked by electrical field stimulation (EFS) caused an elevation in endfoot Ca2+ and vasodilation in brain slices from control animals. However, in brain slices from SAH animals, EFS induced a similar increase in astrocyte endfoot Ca2+ that caused arteriolar constriction rather than vasodilation. Vasoconstriction was observed in approximately 90 % of brain slices from SAH animals in response to EFS, with 40 % exhibiting a sustained vasoconstriction, 30 % exhibiting a transient vasoconstriction (diameter restored within 1 min after EFS), and 20 % responded with a biphasic response (brief vasodilation followed by vasoconstriction). This inversion of neurovascular coupling may play a role in the development of neurological deficits following SAH.
PMCID: PMC3684063  PMID: 22890664
Subarachnoid hemorrhage; neurovascular coupling; cerebral artery; parenchymal arteriole; astrocyte; microcirculation; cortical brain slice; vasospasm
23.  In Vitro and In Vivo Antimalarial Efficacies of Optimized Tetracyclines 
With increasing resistance to existing antimalarials, there is an urgent need to discover new drugs at affordable prices for countries in which malaria is endemic. One approach to the development of new antimalarial drugs is to improve upon existing antimalarial agents, such as the tetracyclines. Tetracyclines exhibit potent, albeit relatively slow, action against malaria parasites, and doxycycline is used for both treatment (with other agents) and prevention of malaria. We synthesized 18 novel 7-position modified tetracycline derivatives and screened them for activity against cultured malaria parasites. Compounds with potent in vitro activity and other favorable drug properties were further tested in a rodent malaria model. Ten compounds inhibited the development of cultured Plasmodium falciparum with a 50% inhibitory concentration (IC50) after 96 h of incubation of <30 nM, demonstrating activity markedly superior to that of doxycycline (IC50 at 96 h of 320 nM). Most compounds showed little mammalian cell cytotoxicity and no evidence of in vitro phototoxicity. In a murine Plasmodium berghei model, 13 compounds demonstrated improved activity relative to that of doxycycline. In summary, 7-position modified tetracyclines offer improved activity against malaria parasites compared to doxycycline. Optimized compounds may allow lower doses for treatment and chemoprophylaxis. If safety margins are adequate, dosing in children, the group at greatest risk for malaria in countries in which it is endemic, may be feasible.
PMCID: PMC3697387  PMID: 23629719
25.  Effects of telephone health mentoring in community-recruited chronic obstructive pulmonary disease on self-management capacity, quality of life and psychological morbidity: a randomised controlled trial 
BMJ Open  2013;3(9):e003097.
To assess benefits of telephone-delivered health mentoring in community-based chronic obstructive pulmonary disease (COPD).
Cluster randomised controlled trial.
Tasmanian general practices: capital city (11), large rural (3), medium rural (1) and small rural (16).
Patients were invited (1207) from general practitioner (GP) databases with COPD diagnosis and/or tiotropium prescription, response rate 49% (586), refused (176) and excluded (criteria: smoking history or previous study, 68). Spirometry testing (342) confirmed moderate or severe COPD in 182 (53%) patients.
By random numbers code, block stratified on location, allocation by sequentially numbered, opaque and sealed envelopes.
Health mentor (HM) group received regular calls to manage illness issues and health behaviours from trained community health nurses using negotiated goal setting: problem solving, decision-making and action planning. Control: usual care (UC) group received GP care plus non-interventional brief phone calls.
Measured at 0, 6 and 12 months, the Short Form 36 (SF-36) and St George’s Respiratory Questionnaire (SGRQ, primary); Partners In Health (PIH) Scale for self-management capacity, Hospital Anxiety and Depression Scale (HADS), Center for Epidemiologic Studies-Depression (CES-D) questionnaire, Post-Traumatic Stress Disorder Checklist, Satisfaction with life and hospital admissions (secondary).
182 participants with COPD (age 68±8 years, 62% moderate COPD and 53% men) were randomised (HM=90 and UC=92). Mixed model regression analysis accounting for clustering, adjusting for age, gender, smoking status and airflow limitation assessed efficacy (regression coefficient, β, reported per 6-month visit). There was no difference in quality of life between groups, but self-management capacity increased in the HM group (PIH overall 0.15, 95% CI 0.03 to 0.29; knowledge domain 0.25, 95% CI 0.00 to 0.50). Anxiety decreased in both groups (HADS A 0.35; 95% CI −0.65 to −0.04) and coping capacity improved (PIH coping 0.15; 95% CI 0.04 to 0.26).
Health mentoring improved self-management capacity but not quality of life compared to regular phone contact, which itself had positive effects where decline is generally expected.
PMCID: PMC3773640  PMID: 24014482
Preventive Medicine; Primary Care

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