A typical procedure for immunoprecipitating a protein (abundance ca 0.5%) synthesized in the wheat germ cell-free system is summarized below. <em>1</em>. Two microliters of 25% SDS are added to 48 microliters of translation reaction mixture, and the sample is heated to <em>1</em>00 degrees for 4 min. 2. Four volumes (i.e., 200 microliters) of dilution buffer at 4 degrees are added to the above sample. Dilution buffer is <em>1</em>.25% Triton X-<em>1</em>00, <em>1</em>90 mM NaCl, 60 mM Tris-HCl, pH 7.4, 6 mM EDTA, <em>1</em>0 units of Trasylol per milliliter. 3. Five microliters of appropriate antisera are added, and the sample is incubated for at least <em>1</em>2 hr at 4 degrees. 4. The sample is spun for 2 min in a microcentrifuge, and the supernatant is transferred to a fresh tube. 5. Thirty microliters of a <em>1</em> : <em>1</em> suspension of protein A-Sepharose CL-4B (<em>1</em>5 microliters of packed beads) are added, and the sample is incubated with end-over-end mixing at room temperature for 2 hr. 6. The Sepharose beads are pelleted by a <em>1</em>0-sec centrifugation in the microcentrifuge, and the supernatant is aspirated. 7. The beads are washed four times in <em>1</em> <em>ml</em>, per wash, of 0.<em>1</em>% Triton X-<em>1</em>00, 0.02% SDS, <em>1</em>50 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, <em>1</em>0 units of Trasylol per milliliter at room temperature with vortexing at each wash. 8. The beads are given a final wash with the above solution not containing detergent, and the supernatant is aspirated as completely as possible with a drawn-out Pasteur pipette. 9. Forty microliters of SDS-gel electrophoresis sample buffer containing 50 mM DTT are added to the beads, and the sample is heated for 4 min in a boiling water bath. <em>1</em>0. Free--SH groups are blocked by adding <em>1</em>0 microliters of <em>1</em>.0 M iodoacetamide in sample buffer and incubating for 45 min at 37 degrees. <em>1</em><em>1</em>. The beads are centrifuged out, and the supernatant is applied to an SDS-polyacrylamide slab gel.

BACKGROUND

Xpert MTB/RIF is a novel automated molecular diagnostic recently endorsed by the World Health Organization. However, performance-related data from high HIV prevalence settings are limited.

OBJECTIVE

The impact of sample-related factors on performance and the significance of Xpert MTB/RIF-positive culture-negative discordance remain unclear.

METHODS

Xpert MTB/RIF was evaluated using single archived spot-sputum samples from 496 South African patients with suspected TB. Mycobacterium tuberculosis culture positivity and phenotypic resistance to rifampicin served as reference standards.

RESULTS

Overall, Xpert MTB/RIF detected 95% (95% confidence interval [CI], 88-98%; 89 of 94) of smear-positive culture-positive cases and the specificity was 94% (9<em>1</em>-96%; 320 of 339). The sensitivity in smear-negative cases was 55% (35-73%; <em>1</em>2 of 22) when the analysis was restricted to <em>1</em> <em>ml</em> of unprocessed sputum and culture time-to-positivity of less than or equal to 28 days. Compared with smear microscopy (n=94), Xpert MTB/RIF detected an additional <em>1</em>7 cases (n=<em>1</em><em>1</em><em>1</em>) representing an <em>1</em>8% (<em>1</em><em>1</em>-27%; <em>1</em><em>1</em><em>1</em> vs. 94) relative increase in the rapid TB case detection rate. Moreover, compared with smear microscopy, the inclusion of Xpert MTB/RIF-positive culture-negative TB cases (ruled-in by an alternative diagnostic method) resulted in the detection of a further <em>1</em>6 cases (n=<em>1</em>27), thus significantly increasing the rapid TB case detection rate to 35% (95% CI, 26-45%; 94 to <em>1</em><em>1</em><em>1</em> vs. 94 to <em>1</em>27; P<0.0<em>1</em>), the overall specificity to 99.<em>1</em>% (97-<em>1</em>00%; 320 of 323; P<0.00<em>1</em>), and sensitivity in smear-negative TB to 60% (P=0.<em>1</em>2). Performance strongly correlated with smear status and culture time-to-positivity. In patients infected with HIV compared with patients uninfected with HIV Xpert MTB/RIF showed a trend to reduced sensitivity (P=0.09) and significantly reduced negative predictive value (P=0.0<em>1</em>). The negative predictive value for rifampicin resistance was 99.4%.

CONCLUSIONS

XpertMTB/RIF outperformed smear microscopy, established a diagnosis in a significant proportion of patients with smear-negative TB, detected many highly likely TB cases missed by culture, and accurately ruled out rifampicin-resistant TB. Sample-specific factors had limited impact on performance. Performance in patients infected with HIV, especially those with advanced immunosuppression, warrants further study.

We studied the effect of prenatal maternal cigarette smoking on the pulmonary function (PF) of 80 healthy infants tested shortly after birth (mean, 4.2 +/- <em>1</em>.9 wk). Mothers' prenatal smoking was measured by: (<em>1</em>) questionnaire reports at each prenatal visit of the number of cigarettes smoked per day, and (2) urine cotinine concentrations (corrected for creatinine) obtained at each visit. Infant PF was assessed by partial expiratory flow-volume curves and helium-dilution measurement of FRC. Forced expiratory flow rates were significantly lower in infants born to smoking mothers, both when unadjusted and after controlling for infant size, age, sex, and passive exposure to environmental tobacco smoke (ETS) between birth and the time of PF testing. Flow at functional residual capacity (VFRC) in infants born to smoking mothers was lower than that found in infants whose mothers did not smoke during pregnancy (74.3 +/- <em>1</em>5.9 versus <em>1</em>50.4 +/- 8.9 <em>ml</em>/s; p = 0.0007). Differences remained significant when flow was corrected for lung size (VFRC/FRC: 0.87 +/- 0.26 versus <em>1</em>.77 +/- 0.<em>1</em>2 s-<em>1</em>; p = 0.0<em>1</em>3). No differences in pulmonary function were evident among infants exposed and unexposed to ETS in the home after stratifying by prenatal exposure status. We conclude that maternal smoking during pregnancy is associated with significant reductions in forced expiratory flow rates in young infants. The results suggest that maternal smoking during pregnancy may impair in utero airway development and/or alter lung elastic properties. We speculate that these effects of maternal prenatal smoking on early levels of forced expiratory flow may be an important factor predisposing infants to the occurrence of wheezing illness later in childhood.

BACKGROUND

Diseases with elevated levels of parathyroid hormone (PTH) such as primary and secondary hyperparathyroidism are associated with increased incidence of cardiovascular disease and death. However, data on the prospective association between circulating PTH levels and cardiovascular mortality in the community are lacking.

RESULTS

The Uppsala Longitudinal Study of Adult Men (ULSAM), a community-based cohort of elderly men (mean age, 7<em>1</em> years; n=958), was used to investigate the association between plasma PTH and cardiovascular mortality. During follow-up (median, 9.7 years), <em>1</em><em>1</em>7 participants died of cardiovascular causes. In Cox proportional-hazards models adjusted for established cardiovascular risk factors (age, systolic blood pressure, diabetes, smoking, body mass index, total cholesterol, high-density lipoprotein cholesterol, antihypertensive treatment, lipid-lowering treatment, and history of cardiovascular disease), higher plasma PTH was associated with higher risk for cardiovascular mortality (hazard ratio for <em>1</em>-SD increase in PTH, <em>1</em>.38; 95% confidence interval, <em>1</em>.<em>1</em>8 to <em>1</em>.60; P<0.00<em>1</em>). This association remained essentially unaltered in participants without previous cardiovascular disease and in participants with normal PTH (<6.8 pmol/L) with no other signs of a disturbed mineral metabolism (normal serum calcium, 2.2 to 2.6 mmol/L; normal glomerular filtration rate, >50 <em>mL</em> . min(-<em>1</em>) . <em>1</em>.73 m(-2) and without vitamin D deficiency, plasma 25-OH vitamin D >37.5 nmol/L). Interestingly, elevated plasma PTH (>5.27 pmol/L) accounted for 20% (95% confidence interval, <em>1</em>0 to 26) of the population-attributable risk proportion for cardiovascular mortality.

CONCLUSIONS

Plasma PTH levels predict cardiovascular mortality in the community, even in individuals with PTH within the normal range. Further studies are warranted to evaluate the clinical implications of measuring PTH in cardiovascular risk prediction and to elucidate whether PTH is a modifiable risk factor.

Hepatitis C virus (HCV) remains a significant threat to the general health of the world's population, and there is a pressing need for the development of new treatments and preventative vaccines. Here, we describe the generation of retrovirus-based pseudoparticles (HCVpp) incorporating a panel of full-length E<em>1</em>E2 clones representative of the major genotypes <em>1</em> through 6, and their application to assess the reactivity and neutralizing capability of antisera and monoclonal antibodies raised against portions of the HCV E2 envelope protein. Rabbit antisera raised against either the first hypervariable region or ectodomain of E2 showed limited and strain specific neutralization. By contrast, the monoclonal antibody (MAb) AP33 demonstrated potent neutralization of infectivity against HCVpp carrying E<em>1</em>E2 representative of all genotypes tested. The concentration of AP33 required to achieve 50% inhibition of infection by HCVpp of diverse genotypes ranged from 0.6 to 32 mug/<em>ml</em>. The epitope recognized by MAb AP33 is linear and highly conserved across different genotypes of HCV. Thus, identification of a broadly neutralizing antibody that recognizes a linear epitope is likely to be of significant benefit to future vaccine and therapeutic antibody development.

BACKGROUND

The problems of adherence to energy restriction in humans are well known.

OBJECTIVE

To compare the feasibility and effectiveness of intermittent continuous energy (IER) with continuous energy restriction (CER) for weight loss, insulin sensitivity and other metabolic disease risk markers.

METHODS

Randomized comparison of a 25% energy restriction as IER (∼ 27<em>1</em>0 kJ/day for 2 days/week) or CER (∼ 6276 kJ/day for 7 days/week) in <em>1</em>07 overweight or obese (mean (± s.d.) body mass index 30.6 (± 5.<em>1</em>) kg m(-2)) premenopausal women observed over a period of 6 months. Weight, anthropometry, biomarkers for breast cancer, diabetes, cardiovascular disease and dementia risk; insulin resistance (HOMA), oxidative stress markers, leptin, adiponectin, insulin-like growth factor (IGF)-<em>1</em> and IGF binding proteins <em>1</em> and 2, androgens, prolactin, inflammatory markers (high sensitivity C-reactive protein and sialic acid), lipids, blood pressure and brain-derived neurotrophic factor were assessed at baseline and after <em>1</em>, 3 and 6 months.

RESULTS

Last observation carried forward analysis showed that IER and CER are equally effective for weight loss: mean (95% confidence interval ) weight change for IER was -6.4 (-7.9 to -4.8) kg vs -5.6 (-6.9 to -4.4) kg for CER (P-value for difference between groups = 0.4). Both groups experienced comparable reductions in leptin, free androgen index, high-sensitivity C-reactive protein, total and LDL cholesterol, triglycerides, blood pressure and increases in sex hormone binding globulin, IGF binding proteins <em>1</em> and 2. Reductions in fasting insulin and insulin resistance were modest in both groups, but greater with IER than with CER; difference between groups for fasting insulin was -<em>1</em>.2 (-<em>1</em>.4 to -<em>1</em>.0) μU <em>ml</em>(-<em>1</em>) and for insulin resistance was -<em>1</em>.2 (-<em>1</em>.5 to -<em>1</em>.0) μU mmol(-<em>1</em>) l(-<em>1</em>) (both P = 0.04).

CONCLUSIONS

IER is as effective as CER with regard to weight loss, insulin sensitivity and other health biomarkers, and may be offered as an alternative equivalent to CER for weight loss and reducing disease risk.

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes >>/=<em>1</em> <em>mL</em>) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (<em>1</em>90 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (5<em>1</em>) of high-aspect ratio microchannels (35 mum width x <em>1</em>50 mum depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near <em>1</em>00% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.

Static muscular contraction has been fir<em>ml</em>y established to reflexly increase cardiovascular and ventilatory function. Although group III and IV fibers with endings in muscle have been shown to comprise the afferent arm of this reflex arc, little is known about the nature of the contraction-induced stimulus causing the activation of these fibers. This stimulus has often been suggested to be a metabolic product of muscular contraction. We have therefore recorded the impulse activity of group III and IV afferents with endings in the triceps surae muscles of barbiturate-anesthetized cats while we injected into the femoral artery substances believed to be metabolic products of muscular contraction. We found that lithium and sodium lactate (400 mM; <em>1</em> <em>ml</em>) had little or no effect on the discharge of group III and IV afferents. Likewise, monobasic sodium phosphate (20 and 400 mM; <em>1</em> <em>ml</em>) and 2-chloroadenosine (50-<em>1</em>00 micrograms) had only trivial effects on the discharge of these afferents. By contrast, lactic acid (25 and 400 mM; <em>1</em> <em>ml</em>) and arachidonic acid (0.5-2.0 mg) caused significant increases in the activity of group III and IV afferents. Most of the excitatory effect of arachidonic acid on the discharge of the afferents was prevented by indomethacin, a cyclooxygenase inhibitor. We conclude that of the substances tested in our experiments, lactic acid and some cyclooxygenase products, such as prostaglandins and thromboxanes, are the most likely to be responsible for any metabolic stimulation of group III and IV afferents during muscular contraction.

OBJECTIVE

We sought to determine whether structured exercise training (ET) improves maximal exercise capacity, left ventricular diastolic function, and quality of life (QoL) in patients with heart failure with preserved ejection fraction (HFpEF).

BACKGROUND

Nearly one-half of patients with heart failure experience HFpEF, but effective therapeutic strategies are sparse.

METHODS

A total of 64 patients (age 65 ± 7 years, 56% female) with HFpEF were prospectively randomized (2:<em>1</em>) to supervised endurance/resistance training in addition to usual care (ET, n = 44) or to usual care alone (UC) (n = 20). The primary endpoint was the change in peak Vo(2) after 3 months. Secondary endpoints included effects on cardiac structure, diastolic function, and QoL.

RESULTS

Peak Vo(2) increased (<em>1</em>6.<em>1</em> ± 4.9 ml/min/kg to <em>1</em>8.7 ± 5.4 ml/min/kg; p < 0.00<em>1</em>) with ET and remained unchanged (<em>1</em>6.7 ± 4.7 ml/min/kg to <em>1</em>6.0 ± 6.0 ml/min/kg; p = NS) with UC. The mean benefit of ET was 3.3 ml/min/kg (95% confidence interval [CI]: <em>1</em>.8 to 4.8, p < 0.00<em>1</em>). E/e' (mean difference of changes: -3.2, 95% CI: -4.3 to -2.<em>1</em>, p < 0.00<em>1</em>) and left atrial volume index (milliliters per square meter) decreased with ET and remained unchanged with UC (-4.0, 95% CI: -5.9 to -2.2, p < 0.00<em>1</em>). The physical functioning score (36-Item Short-Form Health Survey) improved with ET and remained unchanged with UC (<em>1</em>5, 95% CI: 7 to 24, p < 0.00<em>1</em>). The ET-induced decrease of E/e' was associated with 38% gain in peak Vo(2) and 50% of the improvement in physical functioning score.

CONCLUSIONS

Exercise training improves exercise capacity and physical dimensions of QoL in HFpEF. This benefit is associated with atrial reverse remodeling and improved left ventricular diastolic function. (Exercise Training in Diastolic Heart Failure-Pilot Study: A Prospective, Randomised, Controlled Study to Determine the Effects of Physical Training on Exercise Capacity and Quality of Life [Ex-DHF-P]; ISRCTN42524037).

BACKGROUND

Matrix metalloproteinase (MMP) expression is related to blood brain barrier disruption after cerebral ischemia. Moreover, MMP inhibitors reduce hemorrhagic transformation (HT) after embolic ischemia in tissue plasminogen activator (t-PA)-treated animals. We aimed to correlate plasmatic MMP levels with the appearance of intracranial bleeding complications in stroke patients treated with t-PA.

RESULTS

Serial MMP-2 and MMP-9 determinations were performed (ELISA, ng/<em>mL</em>) in 4<em>1</em> strokes involving the middle cerebral artery territory in patients who received t-PA within 3 hours of stroke onset. Blood samples were obtained at baseline (pretreatment) and at <em>1</em>2 and 24 hours after symptom onset. Hemorrhagic events were classified according to CT criteria (petechial hemorrhagic infarctions [HI, <em>1</em> to 2] and large parenchymal hemorrhages [PH, <em>1</em> to 2]). Brain CT scan was obtained at 48 hours or when a neurological worsening occurred. HT was present in 36.5% of the patients (24.4% HI and <em>1</em>2.<em>1</em>% PH). MMP-2 values were unrelated to any subtype of HT. The highest baseline MMP-9 level (normal range <97 ng/<em>mL</em>) corresponded to patients who later developed a PH (PH: 270.2+/-87.8, non-HT: <em>1</em>26.3+/-<em>1</em>27.5, HI: 94.6+/-88.7; P=0.047). A graded response was found between mean baseline MMP-9 levels and the degree of bleeding (HI-<em>1</em>=37.4; HI-2=<em>1</em><em>1</em><em>1</em>.0; PH-<em>1</em>=202.5; PH-2=337.8). Baseline MMP-9 was the most powerful predictor of PH appearance in the multiple logistic regression model (OR= 9.62; CI <em>1</em>.3<em>1</em> to 70.26; P=0.025).

CONCLUSIONS

Baseline MMP-9 level predicts PH appearance after t-PA treatment. Therefore, we suggest that MMP determination may increase the safety profile for thrombolysis and, in the future, anti-MMP drugs might be combined with t-PA to prevent hemorrhagic complications.

The acute inflammatory response is frequently accompanied by serious thrombotic events. We show that C-reactive protein (CRP), an acute-phase reactant that markedly increases its serum concentration in response to inflammatory stimuli, induced monocytes to express tissue factor (TF), a potent procoagulant. Purified human CRP in concentrations commonly achieved in vivo during inflammation (<em>1</em>0 to <em>1</em>00 micrograms/<em>mL</em>) induced a 75-fold increase in TF procoagulant activity (PCA) of human peripheral blood mononuclear cells (PBM), with a parallel increase in TF antigen levels. CRP-induced PCA was completely blocked by a monoclonal antibody against human TF but not by irrelevant murine IgG. Dot blot analysis showed a significant increase of TF mRNA after 4 hours of incubation with CRP, followed by a peak of PCA within 6 and 8 hours. Actinomycin D and cycloheximide blocked CRP-stimulated PCA, suggesting that de novo TF protein synthesis was required. Endotoxin (LPS) contamination of CRP was excluded as the mediator of TF synthesis because: (<em>1</em>) CRP was Limulus assay negative; (2) induction of TF PCA by CRP was not blocked by Polymyxin B, in contrast to LPS-induced PCA; (3) antihuman CRP IgG inhibited CRP-induced PCA, but not LPS-induced PCA; (4) CRP was able to stimulate TF production in LPS-pretreated PBM refractory to additional LPS stimulation; and, (5) unlike LPS, CRP was incapable of inducing TF in human umbilical vein endothelial cells. We suggest that CRP-mediated TF production in monocytes may contribute to the development of disseminated intravascular coagulation and thrombosis in inflammatory states.

BACKGROUND

We evaluated the role of aldosterone as a mediator of renal inflammation and fibrosis in a rat model of aldosterone/salt hypertension using the selective aldosterone blocker, eplerenone.

METHODS

Unnephrectomized, Sprague-Dawley rats were given <em>1</em>% NaCl (salt) to drink and randomized to receive treatment for 28 days: vehicle infusion (control); 0.75 microg/hour aldosterone subcutaneous infusion; or aldosterone infusion + <em>1</em>00 mg/kg/day oral dose of eplerenone. Blood pressure and urinary albumin were measured and kidneys were evaluated histologically. Renal injury, inflammation, and fibrosis were assessed by immunohistochemistry, in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR).

RESULTS

Aldosterone/salt induced severe hypertension compared to controls (220 +/- 4 mm Hg vs. <em>1</em>3<em>1</em> +/- 4 mm Hg, P < 0.05), which was partially attenuated by eplerenone (<em>1</em>79 +/- 4 mm Hg, P < 0.05). In aldosterone/salt treated rats, renal histopathologic evaluation revealed severe vascular and glomerular sclerosis, fibrinoid necrosis and thrombosis, interstitial leukocyte infiltration, and tubular damage and regeneration. Aldosterone/salt increased circulating osteopontin (925.0 +/- 80.2 ng/mL vs. 53.6 +/- 6.3 ng/mL) and albuminuria (75.8 +/- <em>1</em>0.9 mg/24 hours vs. <em>1</em>3.2 +/- 3.0 mg/24 hours) compared to controls and increased expression of proinflammatory molecules. Treatment with eplerenone reduced systemic osteopontin (58.3 +/- 4.2 ng/mL), albuminuria (4<em>1</em>.5 +/- 7.2 mg/24 hours), and proinflammatory gene expression: osteopontin (OPN), monocyte chemoattractant protein-<em>1</em> (MCP-<em>1</em>), interleukin-6 (IL-6), and interleukin-<em>1</em>beta (IL-<em>1</em>beta).

CONCLUSIONS

These findings indicate that aldosterone/salt-induced renal injury and fibrosis has inflammatory components involving macrophage infiltration and cytokine up-regulation. Attenuation of renal damage and inflammation by eplerenone supports the protective effects of aldosterone blockade in hypertensive renal disease.

BACKGROUND

There are few data on the epidemiology of primary HIV-<em>1</em> drug resistance after the roll-out of antiretroviral treatment (ART) in sub-Saharan Africa. We aimed to assess the prevalence of primary resistance in six African countries after ART roll-out and if wider use of ART in sub-Saharan Africa is associated with rising prevalence of drug resistance.

METHODS

We did a cross-sectional study in antiretroviral-naive adults infected with HIV-<em>1</em> who had not started first-line ART, recruited between 2007 and 2009 from <em>1</em><em>1</em> regions in Kenya, Nigeria, South Africa, Uganda, Zambia, and Zimbabwe. We did population-based sequencing of the pol gene on plasma specimens with greater than <em>1</em>000 copies per mL of HIV RNA. We identified drug-resistance mutations with the WHO list for transmitted resistance. The prevalence of sequences containing at least one drug-resistance mutation was calculated accounting for the sampling weights of the sites. We assessed the risk factors of resistance with multilevel logistic regression with random coefficients.

RESULTS

2436 (94.<em>1</em>%) of 2590 participants had a pretreatment genotypic resistance result. <em>1</em>486 participants (57.4%) were women, <em>1</em>575 (60.8%) had WHO clinical stage 3 or 4 disease, and the median CD4 count was <em>1</em>33 cells per μL (IQR 62-204). Overall sample-weighted drug-resistance prevalence was 5.6% (<em>1</em>39 of 2436; 95% CI 4.6-6.7), ranging from <em>1</em>.<em>1</em>% (two of <em>1</em>76; 0.0-2.7) in Pretoria, South Africa, to <em>1</em>2.3% (22 of <em>1</em>79; 7.5-<em>1</em>7.<em>1</em>) in Kampala, Uganda. The pooled prevalence for all three Ugandan sites was <em>1</em><em>1</em>.6% (66 of 570; 8.9-<em>1</em>4.2), compared with 3.5% (73 of <em>1</em>866; 2.5-4.5) for all other sites. Drug class-specific resistance prevalence was 2.5% (54 of 2436; <em>1</em>.8-3.2) for nucleoside reverse-transcriptase inhibitors (NRTIs), 3.3% (83 of 2436; 2.5-4.2) for non-NRTIs (NNRTIs), <em>1</em>.3% (3<em>1</em> of 2436; 0.8-<em>1</em>.8) for protease inhibitors, and <em>1</em>.2% (25 of 2436; 0.7-<em>1</em>.7) for dual-class resistance to NRTIs and NNRTIs. The most common drug-resistance mutations were K<em>1</em>03N (43 [<em>1</em>.8%] of 2436), thymidine analogue mutations (33 [<em>1</em>.6%] of 2436), M<em>1</em>84V (25 [<em>1</em>.2%] of 2436), and Y<em>1</em>8<em>1</em>C/I (<em>1</em>9 [0.7%] of 2436). The odds ratio for drug resistance associated with each additional year since the start of the ART roll-out in a region was <em>1</em>.38 (95% CI <em>1</em>.<em>1</em>3-<em>1</em>.68; p=0.00<em>1</em>).

CONCLUSIONS

The higher prevalence of primary drug resistance in Uganda than in other African countries is probably related to the earlier start of ART roll-out in Uganda. Resistance surveillance and prevention should be prioritised in settings where ART programmes are scaled up.

BACKGROUND

Ministry of Foreign Affairs of the Netherlands.

Uric acid may mediate aspects of the relationship between hypertension and kidney disease via renal vasoconstriction and systemic hypertension. To investigate the relationship between uric acid and subsequent reduced kidney function, limited-access data of <em>1</em>3,338 participants with intact kidney function in two community-based cohorts, the Atherosclerosis Risks in Communities and the Cardiovascular Health Study, were pooled. Mean baseline serum uric acid was 5.9 +/- <em>1</em>.5 mg/dl, mean baseline serum creatinine was 0.9 +/- 0.2 mg/dl, and mean baseline estimated GFR was 90.4 +/- <em>1</em>9.4 <em>ml</em>/min/<em>1</em>.73 m(2). During 8.5 +/- 0.9 yr of follow-up, 7<em>1</em>2 (5.6%) had incident kidney disease defined by GFR decrease >>or=<em>1</em>5 <em>ml</em>/min/<em>1</em>.73 m(2) with final GFR <60 <em>ml</em>/min/<em>1</em>.73 m(2)), while 302 (2.3%) individuals had incident kidney disease defined by creatinine increase >>or=0.4 mg/dl with final serum creatinine>><em>1</em>.4 mg/dl in men and <em>1</em>.2 mg/dl in women). In GFR- and creatinine-based logistic regression models, baseline uric acid level was associated with increased risk for incident kidney disease (odds ratio <em>1</em>.07 [95% confidence interval <em>1</em>.0<em>1</em> to <em>1</em>.<em>1</em>4] and <em>1</em>.<em>1</em><em>1</em> [95% confidence interval <em>1</em>.02 to <em>1</em>.2<em>1</em>] per <em>1</em>-mg/dl increase in uric acid, respectively), after adjustment for age, gender, race, diabetes, systolic BP, hypertension, cardiovascular disease, left ventricular hypertrophy, smoking, alcohol use, education, lipids, albumin, hematocrit, baseline kidney function and cohort; therefore, elevated serum uric acid level is a modest, independent risk factor for incident kidney disease in the general population.

BACKGROUND

Exosomes are released from multiple cell types, contain protein and RNA species, and have been exploited as a novel reservoir for disease biomarker discovery. They can transfer information between cells and may cause pathology, for example, a role for exosomes has been proposed in the pathophysiology of Alzheimer's disease. Although studied in several biofluids, exosomes have not been extensively studied in the cerebrospinal fluid (CSF) from humans. The objective of this study was to determine: <em>1</em>) whether human CSF contains exosomes and 2) the variability in exosomal protein content across individuals.

METHODS

CSF was collected from 5 study participants undergoing thoraco-abdominal aortic aneurysm repair (around 200 - 500 ml per participant) and low-density membrane vesicles were concentrated by ultracentrifugation. The presence of exosomes was determined by western blot for marker proteins, isopycnic centrifugation on a sucrose step gradient and transmission electron microscopy with immuno-labelling. Whole protein profiling was performed using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR).

RESULTS

Flotillin <em>1</em> and tumor susceptibility gene <em>1</em>0<em>1</em> (TSG<em>1</em>0<em>1</em>), two exosomal marker proteins, were identified in the ultracentrifugation pellet using western blot. These markers localized to a density consistent with exosomes following isopycnic centrifugation. Transmission electron microscopy visualized structures consistent with exosomes in size and appearance that labelled positive for flotillin <em>1</em>. Therefore, the pellet that resulted from ultracentrifugation of human CSF contained exosomes. FT-ICR profiling of this pellet was performed and 84-<em>1</em>6<em>1</em> ions were detected per study participant. Around one third of these ions were only present in a single study participant and one third were detected in all five. With regard to ion quantity, the median coefficient of variation was 8<em>1</em>% for ions detected in two or more samples.

CONCLUSIONS

Exosomes were identified in human CSF and their proteome is a potential new reservoir for biomarker discovery in neurological disorders such as Alzheimer's disease. However, techniques used to concentrate exosomes from CSF need refinement to reduce variability. In this study we used relatively large starting volumes of human CSF, future studies will focus on exosome isolation from smaller 'real life' clinical samples; a key challenge in the development of exosomes as translational tools.

BACKGROUND

There are no validated measures of adherence to HIV antiretroviral therapy in resource-poor settings. Such measures are essential to understand the unique barriers to adherence as access to HIV antiretroviral therapy expands.

METHODS

We assessed correspondence between multiple measures of adherence and viral load suppression in 34 patients purchasing generic Triomune antiretroviral therapy (coformulated stavudine, lamivudine, and nevirapine; CIPLA, Ltd., Mumbai, India) in Kampala, Uganda. Measures included 3-day patient self-report, 30-day visual analog scale, electronic medication monitoring, and unannounced home pill count. HIV-<em>1</em> load was determined at baseline and <em>1</em>2 weeks.

RESULTS

Mean adherence was 9<em>1</em>%-94% by all measures. Seventy-six percent of subjects had a viral load of <400 copies/mL at <em>1</em>2 weeks. All measures were closely correlated with each other (R = 0.77-0.89). Each measure was also significantly associated with <em>1</em>2-week HIV load. There was no significant difference between patient-reported and objective measures of adherence.

CONCLUSIONS

This sample of patients purchasing generic HIV antiretroviral therapy has among the highest measured adherence reported to date. Patient-reported measures were closely associated with objective measures. The relative ease of administration of the 30-day visual analog scale suggests that this may be the preferred method to assess adherence in resource-poor settings.

OBJECTIVE

Full length keratin-<em>1</em>8 (FL-K<em>1</em>8) and High Mobility Group Box-<em>1</em> (HMGB<em>1</em>) represent circulating indicators of necrosis during acetaminophen (APAP) hepatotoxicity in vivo. In addition, the caspase-cleaved fragment of K<em>1</em>8 (cK<em>1</em>8) and hyper-acetylated HMGB<em>1</em> represent serum indicators of apoptosis and immune cell activation, respectively. The study aim was to assess their mechanistic utility to establish the balance between apoptosis, necrosis, and immune cell activation throughout the time course of clinical APAP hepatotoxicity.

METHODS

HMGB<em>1</em> (total, acetylated) and K<em>1</em>8 (apoptotic, necrotic) were identified and quantified by novel LC-MS/MS assays in APAP overdose patients (n=78).

RESULTS

HMGB<em>1</em> (total; <em>1</em>5.4±<em>1</em>.9ng/ml, p<0.0<em>1</em>, acetylated; 5.4±2.6ng/ml, p<0.00<em>1</em>), cK<em>1</em>8 (5649.8±72<em>1</em>.0U/L, p<0.0<em>1</em>), and FL-K<em>1</em>8 (54770.2±67<em>1</em>7.0U/L, p<0.005) were elevated in the sera of APAP overdose patients with liver injury compared to overdose patients without liver injury and healthy volunteers. HMGB<em>1</em> and FL-K<em>1</em>8 correlated with alanine aminotransferase (ALT) activity (R(2)=0.60 and 0.58, respectively, p<0.000<em>1</em>) and prothrombin time (R(2)=0.62 and 0.7<em>1</em>, respectively, p<0.000<em>1</em>). Increased total and acetylated HMGB<em>1</em> and FL-K<em>1</em>8 were associated with worse prognosis (King's College Criteria) or patients that died/required liver transplant compared to spontaneous survivors (all p<0.05-0.00<em>1</em>), a finding not reflected by ALT and supported by ROC analysis. Acetylated HMGB<em>1</em> was a better predictor of outcome than the other markers of cell death.

CONCLUSIONS

K<em>1</em>8 and HMGB<em>1</em> represent blood-based tools to investigate the cell death balance clinical APAP hepatotoxicity. Activation of the immune response was seen later in the time course as shown by the distinct profile of acetylated HMGB<em>1</em> and was associated with worse outcome.

Experimental animal models suggest that uric acid might have a pathogenic role in the early development of primary hypertension. We hypothesized that serum uric acid is correlated with blood pressure in children with new-onset, untreated, primary hypertension. We evaluated <em>1</em>25 consecutive children referred to the Baylor Pediatric Renal Program for evaluation of hypertension. None of the subjects had previously been evaluated or treated for hypertension. The children ranged in age from 6 to <em>1</em>8 years (mean, <em>1</em>3.4+/-3.3) and had normal renal function (creatinine clearance >80 <em>mL</em> x min(-<em>1</em>) x <em>1</em>.73 m(-2)). Sixty-three children had primary hypertension, 40 had secondary hypertension, and 22 had white-coat hypertension. Forty controls with normal blood pressure were recruited from the renal clinic. Uric acid levels were directly correlated with systolic (r=0.80, P=0.0002) and diastolic (r=0.66, P=0.0006) blood pressure in controls and in subjects with primary hypertension and were independent of renal function. Serum uric acid concentrations >5.5 mg/dL were found in 89% of subjects with primary hypertension, in 30% with secondary hypertension, in 0% with white-coat hypertension, and in 0% of controls. We conclude that serum uric acid is directly correlated with blood pressure in untreated children and that a serum uric acid value >5.5 mg/dL in an adolescent being evaluated for hypertension strongly suggests primary hypertension as opposed to white-coat or secondary hypertension. These results are consistent with the hypothesis that uric acid might have a role in the early pathogenesis of primary hypertension.

The CLSI established clinical breakpoints (CBPs) for caspofungin (CSF), micafungin (MCF) and anidulafungin (ANF) versus Candida. The same CBP (susceptible (S): MIC ≤ 2 mcg/<em>ml</em>; non-S: MIC>> 2 mcg/<em>ml</em>) was applied to all echinocandins and species. More data now allow reassessment of these CBPs. We examined cases of echinocandin failure where both MICs and fks mutations were assessed; wild type (WT) MICs and epidemiological cutoff values (ECVs) for a large Candida collection; molecular analysis of fks hotspots for Candida with known MICs; and pharmacokinetic and pharmacodynamic (PK/PD) data. We applied these findings to propose new species-specific CBPs for echinocandins and Candida. Of <em>1</em>8 candidiasis cases refractory to echinocandins and with fks mutations, 28% (CSF), 58% (ANF) and 66% (MCF) had MICs in the S category using CBP of ≤ 2 mcg/<em>ml</em>, while 0-8% would be S using CBP of ≤ 0.25 mcg/<em>ml</em>. WT MIC distributions revealed ECV ranges of 0.03-0.25 mcg/<em>ml</em> for all major species except C. parapsilosis (<em>1</em>-4 mcg/<em>ml</em>) and C. guilliermondii (4-<em>1</em>6 mcg/<em>ml</em>). Among Candida tested for fks mutations, only <em>1</em>5.7-45.<em>1</em>% of 5<em>1</em> mutants were detected using the CBP for NS of >2 mcg/<em>ml</em>. In contrast, a cutoff of >0.25 mcg/<em>ml</em> for C. albicans, C. tropicalis, C. krusei, and C. dubliniensis detected 85.6% (MCF) to 95.2% (CSF) of 2<em>1</em> mutant strains. Likewise, a cutoff of >0.<em>1</em>2 mcg/<em>ml</em> for ANF and CSF and of >0.06 mcg/<em>ml</em> for MCF detected 93% (ANF) to 97% (CSF, MCF) of 30 mutant strains of C. glabrata. These data, combined with PK/PD considerations, support CBPs of ≤ 0.25 mcg/<em>ml</em> (S), 0.5 mcg/<em>ml</em> (I), ≥ <em>1</em> (R) for CSF/MCF/ANF and C. albicans, C. tropicalis and C. krusei and ≤ 2 mcg/<em>ml</em> (S), 4 mcg/<em>ml</em> (I), and ≥ 8 mcg/<em>ml</em> (R) for these agents and C. parapsilosis. The CBPs for ANF and CSF and C. glabrata are ≤ 0.<em>1</em>2 mcg/<em>ml</em> (S), 0.25 mcg/<em>ml</em> (I), and ≥ 0.5 mcg/<em>ml</em> (R), whereas those for MCF are ≤ 0.06 mcg/<em>ml</em> (S), 0.<em>1</em>2 mcg/<em>ml</em> (I), and ≥ 0.25 mcg/<em>ml</em> (R). New, species-specific CBPs for Candida and the echinocandins are more sensitive to detect emerging resistance associated with fks mutations, and better able to predict risk for clinical failure.

In this chapter we describe in details the permeabilized cell and skinned fiber techniques and their applications for studies of mitochondrial function in vivo. The experience of more than <em>1</em>0 years of research in four countries is summarized. The use of saponin in very low concentration (50-<em>1</em>00 microg/<em>ml</em>) for permeabilisation of the sarcolemma leaves all intracellular structures, including mitochondria, completely intact. The intactness of mitochondrial function in these skinned muscle fibers is demonstrated in this work by multiple methods, such as NADH and flavoprotein fluorescence studies, fluorescence imaging, confocal immunofluorescence microscopy and respiratory analysis. Permeabilized cell and skinned fiber techniques have several very significant advantages for studies of mitochondrial function, in comparison with the traditional methods of use of isolated mitochondria: (<em>1</em>) very small tissue samples are required; (2) all cellular population of mitochondria can be investigated; (3) most important, however, is that mitochondria are studied in their natural surrounding. The results of research by using this method show the existence of several new phenomenon--tissue dependence of the mechanism of regulation of mitochondrial respiration, and activation of respiration by selective proteolysis. These phenomena are explained by interaction of mitochondria with other cellular structures in vivo. The details of experimental studies with use of these techniques and problems of kinetic analysis of the results are discussed. Examples of large-scale clinical application of these methods are given.

BACKGROUND

Whether the addition of radiation therapy (RT) improves overall survival in men with locally advanced prostate cancer managed with androgen deprivation therapy (ADT) is unclear. Our aim was to compare outcomes in such patients with locally advanced prostate cancer.

METHODS

Patients with: locally advanced (T3 or T4) prostate cancer (n=1057); or organ-confined disease (T2) with either a prostate-specific antigen (PSA) concentration more than 40 ng/mL (n=119) or PSA concentration more than 20 ng/mL and a Gleason score of 8 or higher (n=25), were randomly assigned (done centrally with stratification and dynamic minimisation, not masked) to receive lifelong ADT and RT (65-69 Gy to the prostate and seminal vesicles, 45 Gy to the pelvic nodes). The primary endpoint was overall survival. The results presented here are of an interim analysis planned for when two-thirds of the events for the final analysis were recorded. All efficacy analyses were done by intention to treat and were based on data from all patients. This trial is registered at controlledtrials.com as ISRCTN24991896 and Clinicaltrials.gov as NCT00002633.

RESULTS

Between 1995 and 2005, 1205 patients were randomly assigned (602 in the ADT only group and 603 in the ADT and RT group); median follow-up was 6·0 years (IQR 4·4-8·0). At the time of analysis, a total of 320 patients had died, 175 in the ADT only group and 145 in the ADT and RT group. The addition of RT to ADT improved overall survival at 7 years (74%, 95% CI 70-78 vs 66%, 60-70; hazard ratio [HR] 0·77, 95% CI 0·61-0·98, p=0·033). Both toxicity and health-related quality-of-life results showed a small effect of RT on late gastrointestinal toxicity (rectal bleeding grade >3, three patients (0·5%) in the ADT only group, two (0·3%) in the ADT and RT group; diarrhoea grade >3, four patients (0·7%) vs eight (1·3%); urinary toxicity grade >3, 14 patients (2·3%) in both groups).

CONCLUSIONS

The benefits of combined modality treatment--ADT and RT--should be discussed with all patients with locally advanced prostate cancer.

BACKGROUND

Canadian Cancer Society Research Institute, US National Cancer Institute, and UK Medical Research Council.

OBJECTIVE

We previously showed that brief intermittent ischemia applied during the onset of reperfusion (i.e., postconditioning) is cardioprotective in a canine model of ischemia-reperfusion. This study tested the hypothesis that the early minutes of reperfusion (R) during which postconditioning (Post-con) is applied are critical to its cardioprotection.

METHODS

In anesthetized open-chest rats, the left coronary artery (LCA) was occluded for 30 min and reperfused for 3 h. All rats were randomly divided into six groups: Control (n=8): no intervention at R; Ischemic preconditioning (IPC) (n=8): the LCA was occluded for 5 min followed by <em>1</em>0 min of R before the index occlusion; Post-con <em>1</em> (n=8): after LCA occlusion, three cycles of <em>1</em>0 s R followed by <em>1</em>0 s LCA re-occlusion were applied during the first minute of R; Post-con 2 (n=8): Six cycles of <em>1</em>0 s R and <em>1</em>0 s re-occlusion were applied during the first 2 min of R; Delayed Post-con (n=8): the ligature was loosened for full reflow for the first minute of R, after which the three-cycle Post-con algorithm was applied; Sham (n=6): the surgical procedure was identical to other groups, but the LCA ligature was not ligated.

RESULTS

Infarct size (TTC staining) was 23% smaller in Post-con <em>1</em> (40+/-2%*) than in Control (52+/-3%), confirmed by plasma creatine kinase activity (<em>1</em>8+/-2* vs. 46+/-6 IU/g protein). There was no further reduction in infarct size with 6 cycles of Post-con (40+/-2.9%, p>0.05 vs. Post-con <em>1</em>). Meanwhile, infarct size reduction was significantly greater in the IPC group (<em>1</em>7+/-3%) than in Post-con<em>1</em> (p<0.0<em>1</em>). The plasma lipid peroxidation product malondialdehyde (MDA, microM/ml) was less after R in IPC and Post-con <em>1</em> (0.8+/-0.07* and 0.8+/-0.06*) vs. Control (<em>1</em>.2<em>1</em>+/-0.08), consistent with a visual decrease in superoxide anion generation (dihydroethidium staining) in the AAR myocardium after 3 h of reperfusion. Neutrophil accumulation (myeloperoxidase activity, MPO, U/<em>1</em>00 g tissue) in the AAR was less in IPC (<em>1</em>.4+/-0.3*) and Post-con <em>1</em> (2.5+/-0.3*) vs. Control (5.5+/-0.6). The reductions in infarct size, creatine kinase, MDA and DHE staining were lost with delayed Post-con, while MPO activity remained lower than in Control (3.2+/-0.4*).

CONCLUSIONS

(<em>1</em>) Post-con at onset of R reduces myocardial injury; (2) cardioprotection may be mediated, in part, by inhibiting oxidant generation and oxidant mediated injury; (3) the first minute of R in the rat model is critical to cardioprotection by Post-con; and (4) cardioprotection by Post-con may be independent of neutrophil accumulation in AAR. *p<0.05 Post-con vs. Control.

BACKGROUND

Little is known about the efficacy and safety of renal artery stenting in patients with atherosclerotic renal artery stenosis (ARAS) and impaired renal function.

OBJECTIVE

To determine the efficacy and safety of stent placement in patients with ARAS and impaired renal function.

METHODS

Randomized clinical trial. Randomization was centralized and computer generated, and allocation was assigned by e-mail. Patients, providers, and persons who assessed outcomes were not blinded to treatment assignment.

METHODS

10 European medical centers.

METHODS

140 patients with creatinine clearance less than 80 mL/min per 1.73 m(2) and ARAS of 50% or greater.

METHODS

Stent placement and medical treatment (64 patients) or medical treatment only (76 patients). Medical treatment consisted of antihypertensive treatment, a statin, and aspirin.

METHODS

The primary end point was a 20% or greater decrease in creatinine clearance. Secondary end points included safety and cardiovascular morbidity and mortality.

RESULTS

Forty-six of 64 patients assigned to stent placement had the procedure. Ten of the 64 patients (16%) in the stent placement group and 16 patients (22%) in the medication group reached the primary end point (hazard ratio, 0.73 [95% CI, 0.33 to 1.61]). Serious complications occurred in the stent group, including 2 procedure-related deaths (3%), 1 late death secondary to an infected hematoma, and 1 patient who required dialysis secondary to cholesterol embolism. The groups did not differ for other secondary end points.

CONCLUSIONS

Many patients were falsely identified as having renal artery stenosis greater than 50% by noninvasive imaging and did not ultimately require stenting.

CONCLUSIONS

Stent placement with medical treatment had no clear effect on progression of impaired renal function but led to a small number of significant procedure-related complications. The study findings favor a conservative approach to patients with ARAS, focused on cardiovascular risk factor management and avoiding stenting.

The mode of interaction of the polycationic aminoglycoside antibiotics with the surface of Pseudomonas aeruginosa cells was studied with the hydrophobic fluorescent probe <em>1</em>-N-phenylnaphthylamine (NPN). The addition of the aminoglycoside gentamicin to intact cells in the presence of NPN led to a shift in the fluorescence emission maximum from 460 to 420 nm. At the same time the NPN fluorescence intensity increased fourfold. Gentamicin caused no such effects when added to outer membrane vesicles, suggesting that the increased fluorescence resulted from the interaction of gentamicin with intact cells. Gentamicin-promoted NPN uptake was inhibited by the divalent cations Mg2+ and Ca2+, but occurred in the absence of gentamicin transport across the inner membrane. Low concentrations of gentamicin (2 micrograms/<em>ml</em>) caused NPN fluorescence to increase over a period of 4 min in a sigmoidal fashion. At higher concentrations (50 micrograms/<em>ml</em>) the increase occurred within a few seconds. The final fluorescence intensity was almost independent of the gentamicin concentration. A centrifugation technique was used to demonstrate that gentamicin caused actual uptake of NPN from the supernatant. The initial rate of NPN uptake varied according to the gentamicin concentration in a sigmoidal fashion. Similar data were obtained for seven other aminoglycoside antibiotics. The data, when reanalyzed as a Hill plot, gave a series of lines with a mean slope (the Hill number) of 2.26 +/- 0.26, suggesting that the interaction of aminoglycosides with the cell surface to permeabilize it to NPN involved at least three sites and demonstrated positive cooperativity. There was a statistically significant relationship between the pseudoassociation constant K, from the Hill plots and the minimal inhibitory concentrations for the eight antibiotics. These results are consistent with the concept that aminoglycosides interact as a divalent cation binding site on the P. aeruginosa outer membrane and permeabilize it to the hydrophobic prove NPN.