BACKGROUND

Transmission of HIV-<em>1</em> is predominantly by heterosexual contact in sub-Saharan Africa, where sexually transmitted diseases (STDs) are also common. Epidemiological studies suggest that STDs facilitate transmission of HIV-<em>1</em>, but the biological mechanism remains unclear. We investigated the hypothesis that STDs increase the likelihood of transmission of HIV-<em>1</em> through increased concentration of the virus in semen.

METHODS

HIV-<em>1</em> RNA concentrations were measured in seminal and blood plasma from <em>1</em>35 HIV-<em>1</em>-seropositive men in Malawi; 86 had urethritis and 49 controls did not have urethritis. Men with urethritis received antibiotic treatment according to the guidelines of the Malawian STD Advisory Committee. Samples were analysed at baseline and at week <em>1</em> and week 2 after antibiotic therapy in urethritis patients, and at baseline and week 2 in the control group.

RESULTS

HIV-<em>1</em>-seropositive men with urethritis had HIV-<em>1</em> RNA concentrations in seminal plasma eight times higher than those in seropositive men without urethritis (<em>1</em>2.4 vs <em>1</em>.5<em>1</em> x <em>1</em>0(4) copies/mL, p = 0.035), despite similar CD4 counts and concentrations of blood plasma viral RNA. Gonorrhoea was associated with the greatest concentration of HIV-<em>1</em> in semen (<em>1</em>5.8 x <em>1</em>0(4) copies/mL). After the urethritis patients received antimicrobial therapy directed against STDs, the concentration of HIV-<em>1</em> RNA in semen decreased significantly (from <em>1</em>2.4 x <em>1</em>0(4) copies/mL to 8.9<em>1</em> x <em>1</em>0(4) copies/mL at <em>1</em> week [p = 0.03] and 4.<em>1</em>2 x <em>1</em>0(4) copies/mL at 2 weeks [p = 0.000<em>1</em>]). Blood plasma viral RNA concentrations did not change. There was no significant change in seminal plasma HIV-<em>1</em> RNA concentrations during the 2-week period in the control group (p = 0.42<em>1</em>).

CONCLUSIONS

These results suggest that urethritis increases the infectiousness of men with HIV-<em>1</em> infection. HIV-<em>1</em>-control programmes, which include detection and treatment of STDs in patients already infected with HIV-<em>1</em>, may help to curb the epidemic. Targeting of gonococcal urethritis may be a particularly effective strategy.

We attempted to find a relationship between the microbiological properties of bloodstream isolates of methicillin-resistant Staphylococcus aureus (MRSA) and the efficacy of vancomycin in the treatment of bacteremia. Vancomycin susceptibility testing was performed, and bactericidal activity was determined for 30 isolates from 30 different patients with MRSA bacteremia for whom clinical and microbiological outcome data were available. The majority of these patients had been previously enrolled in multicenter prospective studies of MRSA bacteremia refractory to conventional vancomycin therapy. Logistic regression found a statistically significant relationship between treatment success with vancomycin and decreases in both vancomycin MICs (< or =0.5 microg/<em>ml</em> versus <em>1</em>.0 to 2.0 microg/<em>ml</em>; P = 0.02) and degree of killing (reduction in log(<em>1</em>0) CFU/milliliter) by vancomycin over 72 h of incubation in vitro (P = 0.03). For MRSA isolates with vancomycin MICs < or = 0.5 microg/<em>ml</em>, vancomycin was 55.6% successful in the treatment of bacteremia whereas vancomycin was only 9.5% effective in cases in which vancomycin MICs for MRSA were <em>1</em> to 2 microg/<em>ml</em>. Patients with MRSA that was more effectively killed at 72 h by vancomycin in vitro had a higher clinical success rate with vancomycin therapy in the treatment of bacteremia (log(<em>1</em>0) < 4.7<em>1</em> [n = 9], 0%; log(<em>1</em>0) 4.7<em>1</em> to 6.26 [n = <em>1</em>3], 23.<em>1</em>%; log(<em>1</em>0)>> 6.27 [n = 8], 50%). We conclude that a significant risk for vancomycin treatment failure in MRSA bacteremia begins to emerge with increasing vancomycin MICs well within the susceptible range. Elucidating the mechanisms involved in intermediate-level glycopeptide resistance in S. aureus should begin by examining bacteria that begin to show changes in vancomycin susceptibility before the development of obvious resistance. Prognostic information for vancomycin treatment outcome in MRSA bacteremia may also be obtained by testing the in vitro bactericidal potency of vancomycin.

The accurate quantitation of high density lipoproteins has recently assumed greater importance in view of studies suggesting their negative correlation with coronary heart disease. High density lipoproteins may be estimated by measuring cholesterol in the plasma fraction of d>> <em>1</em>.063 g/<em>ml</em>. A more practical approach is the specific precipitation of apolipoprotein B (apoB)-containing lipoproteins by sulfated polysaccharides and divalent cations, heparin-Mn(2+) being the most commonly used combination. The present heparin-Mn(2+) procedure was found to be reasonably specific and not often subject to large errors; however, 9% (primarily hypertriglyceridemic samples) of the 966 plasma samples treated with heparin-Mn(2+) had obvious supernatant turbidity, indicating incomplete sedimentation of apoB-associated lipoproteins. Furthermore, 48% of the nonturbid supernates contained more than <em>1</em> mg/dl (mean 2.5 mg/dl) of apoB-associated cholesterol when measured by a radial immunodiffusion procedure, indicating slight overestimation of HDL cholesterol. Determination of the extent of the unprecipitated apoB-associated lipoproteins by sensitive radioimmunoassay and of the amount of precipitated high density lipoprotein by radial immunodiffusion assay of apolipoproteins A-I and A-II at various heparin and Mn(2+) concentrations indicated that the usual heparin level (approximately <em>1</em>.3 mg/<em>ml</em>) was adequate. However, a twofold increase in Mn(2+) concentration to 0.092 M improved precipitation of the apoB-associated lipoproteins without excessive precipitation of high density lipoprotein from plasma. This increased Mn(2+) level also provided improved sedimentation of the apoB-associated lipoproteins from hypertriglyceridemic plasma. Additional observations suggested that, for convenience, the heparin and Mn(2+) can be added simultaneously as a combined reagent, that samples can be incubated for <em>1</em>0 minutes at room temperature before centrifugation, and that turbid supernates from hypertriglyceridemic samples can usually be made free of apoB-associated lipoproteins by centrifugation at <em>1</em>2,000 g for <em>1</em>0 minutes.

An inhibitory activity for an erythrocyte in termediate bearing the properdin (P)-stabilized amplification C3 convertase, PC3bBb, was recognized in whole normal human serum and separated from C3b inactivator by its distinct physicochemical and functional characteristics. The inhibitory activity was found to reside in a protein that was purified to homogeneity and elicited a monospecific antibody in a rabbit. This protein was identified as beta<em>1</em>H and found to have a serum concentration of 5<em>1</em>6 +/- 89 mug/<em>ml</em> (mean +/- <em>1</em> SD). beta<em>1</em>H produced a dose related, first-order loss of convertase function and release of <em>1</em>25I-Bb from the P-stabilized intermediate, indicating a mechanism of action by decay-dissociation of Bb from the complex, PC3bBb. beta<em>1</em>H exhibited only a limited capacity to accelerate decay of C3bBb sites stabilized with C3 nephritic factor or to release <em>1</em>25I-Bb from such sites. Amplification of C3 cleavage by C3bBb may well determine whether initial complement activation by the classical or alternative activating sequence is beneficial or detrimental to the host. Regulation of this amplifying function is now recognized to occur at at least three steps: intrinsic decay which reflects the inherent lability of the C3bBb convertase; extrinsic decay-dissociation of Bb which is mediated by the effect of beta<em>1</em>H; and inactivation of exposed C3b by C3b inactivator. The stabilization of C3bBb by activated properdin minimizes intrinsic decay and protects C3b in the bimolecular complex from C3b inactivator. beta<em>1</em>H restores control of the system by decay-dissociation of the bimolecular complex, therby exposing C3b to C3b inactivator whose irreversible action prevents regeneration of the convertase at that site.

Phylogenetic bootstrapping (BS) is a standard technique for inferring confidence values on phylogenetic trees that is based on reconstructing many trees from minor variations of the input data, trees called replicates. BS is used with all phylogenetic reconstruction approaches, but we focus here on one of the most popular, maximum likelihood (<em>ML</em>). Because <em>ML</em> inference is so computationally demanding, it has proved too expensive to date to assess the impact of the number of replicates used in BS on the relative accuracy of the support values. For the same reason, a rather small number (typically <em>1</em>00) of BS replicates are computed in real-world studies. Stamatakis et al. recently introduced a BS algorithm that is <em>1</em> to 2 orders of magnitude faster than previous techniques, while yielding qualitatively comparable support values, making an experimental study possible. In this article, we propose stopping criteria--that is, thresholds computed at runtime to determine when enough replicates have been generated--and we report on the first large-scale experimental study to assess the effect of the number of replicates on the quality of support values, including the performance of our proposed criteria. We run our tests on <em>1</em>7 diverse real-world DNA--single-gene as well as multi-gene--datasets, which include <em>1</em>25-2,554 taxa. We find that our stopping criteria typically stop computations after <em>1</em>00-500 replicates (although the most conservative criterion may continue for several thousand replicates) while producing support values that correlate at better than 99.5% with the reference values on the best <em>ML</em> trees. Significantly, we also find that the stopping criteria can recommend very different numbers of replicates for different datasets of comparable sizes. Our results are thus twofold: (i) they give the first experimental assessment of the effect of the number of BS replicates on the quality of support values returned through BS, and (ii) they validate our proposals for stopping criteria. Practitioners will no longer have to enter a guess nor worry about the quality of support values; moreover, with most counts of replicates in the <em>1</em>00-500 range, robust BS under <em>ML</em> inference becomes computationally practical for most datasets. The complete test suite is available at http://lcbb.epfl.ch/BS.tar.bz2, and BS with our stopping criteria is included in the latest release of RAx<em>ML</em> v7.2.5, available at http://wwwkramer.in.tum.de/exelixis/software.html.

Evidence has emerged that mesenchymal stem cells (MSCs) represent a promising population for supporting new clinical concepts in cellular therapy. However, attempts to isolate MSCs from umbilical cord blood (UCB) of full-term deliveries have previously either failed or been characterized by a low yield. We investigated whether cells with MSC characteristics and multi-lineage differentiation potential can be cultivated from UCB of healthy newborns and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria. Using optimized isolation and culture conditions, in up to 63% of 59 low-volume UCB units, cells showing a characteristic mesenchymal morphology and immune phenotype (MSC-like cells) were isolated. These were similar to control MSCs from adult bone marrow (BM). The frequency of MSC-like cells ranged from 0 to 2.3 clones per <em>1</em> x <em>1</em>0(8) mononuclear cells (MNCs). The cell clones proliferated extensively with at least 20 population doublings within eight passages. In addition, osteogenic and chondrogenic differentiation demonstrated a multi-lineage capacity comparable with BM MSCs. However, in contrast to MSCs, MSC-like cells showed a reduced sensitivity to undergo adipogenic differentiation. Crucial points to isolate MSC-like cells from UCB were a time from collection to isolation of less than <em>1</em>5 hours, a net volume of more than 33 <em>ml</em>, and an MNC count of more than <em>1</em> x <em>1</em>0(8) MNCs. Because MSC-like cells can be isolated at high efficacy from full-term UCB donations, we regard UCB as an additional stem cell source for experimental and potentially clinical purposes.

OBJECTIVE

Hyperuricemia is associated with hypertension, inflammation, renal disease progression, and cardiovascular disease. However, no data are available regarding the effect of allopurinol in patients with chronic kidney disease.

METHODS

We conducted a prospective, randomized trial of <em>1</em><em>1</em>3 patients with estimated GFR (eGFR) <60 <em>ml</em>/min. Patients were rando<em>ml</em>y assigned to treatment with allopurinol <em>1</em>00 mg/d (n = 57) or to continue the usual therapy (n = 56). Clinical, biochemical, and inflammatory parameters were measured at baseline and at 6, <em>1</em>2, and 24 months of treatment. The objectives of study were: (<em>1</em>) renal disease progression; (2) cardiovascular events; and (3) hospitalizations of any causes.

RESULTS

Serum uric acid and C-reactive protein levels were significantly decreased in subjects treated with allopurinol. In the control group, eGFR decreased 3.3 +/- <em>1</em>.2 <em>ml</em>/min per <em>1</em>.73 m(2), and in the allopurinol group, eGFR increased <em>1</em>.3 +/- <em>1</em>.3 <em>ml</em>/min per <em>1</em>.73 m(2) after 24 months. Allopurinol treatment slowed down renal disease progression independently of age, gender, diabetes, C-reactive protein, albuminuria, and renin-angiotensin system blockers use. After a mean follow-up time of 23.4 +/- 7.8 months, 22 patients suffered a cardiovascular event. Diabetes mellitus, previous coronary heart disease, and C-reactive protein levels increased cardiovascular risk. Allopurinol treatment reduces risk of cardiovascular events in 7<em>1</em>% compared with standard therapy.

CONCLUSIONS

Allopurinol decreases C-reactive protein and slows down the progression of renal disease in patients with chronic kidney disease. In addition, allopurinol reduces cardiovascular and hospitalization risk in these subjects.

<strong class="sub-title"> Background: </strong> This is the first randomised controlled trial for assessment of the immunogenicity and safety of a candidate non-replicating adenovirus type-5 (Ad5)-vectored COVID-<em>1</em>9 vaccine, aiming to determine an appropriate dose of the candidate vaccine for an efficacy study.

<strong class="sub-title"> Methods: </strong> This randomised, double-blind, placebo-controlled, phase 2 trial of the Ad5-vectored COVID-<em>1</em>9 vaccine was done in a single centre in Wuhan, China. Healthy adults aged <em>1</em>8 years or older, who were HIV-negative and previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-free, were eligible to participate and were randomly assigned to receive the vaccine at a dose of <em>1</em> × <em>1</em>0^{<em>1</em><em>1</em>} viral particles per <em>mL</em> or 5 × <em>1</em>0^<em>1</em>0 viral particles per <em>mL</em>, or placebo. Investigators allocated participants at a ratio of 2:<em>1</em>:<em>1</em> to receive a single injection intramuscularly in the arm. The randomisation list (block size 4) was generated by an independent statistician. Participants, investigators, and staff undertaking laboratory analyses were masked to group allocation. The primary endpoints for immunogenicity were the geometric mean titres (GMTs) of specific ELISA antibody responses to the receptor binding domain (RBD) and neutralising antibody responses at day 28. The primary endpoint for safety evaluation was the incidence of adverse reactions within <em>1</em>4 days. All recruited participants who received at least one dose were included in the primary and safety analyses. This study is registered with ClinicalTrials.gov, <a href="http://clinicaltrials.gov/show/NCT0434<em>1</em>389" title="See in ClinicalTrials.gov">NCT0434<em>1</em>389</a>.

<strong class="sub-title"> Findings: </strong> 603 volunteers were recruited and screened for eligibility between April <em>1</em><em>1</em> and <em>1</em>6, 2020. 508 eligible participants (50% male; mean age 39·7 years, SD <em>1</em>2·5) consented to participate in the trial and were randomly assigned to receive the vaccine (<em>1</em> × <em>1</em>0^{<em>1</em><em>1</em>} viral particles n=253; 5 × <em>1</em>0^<em>1</em>0 viral particles n=<em>1</em>29) or placebo (n=<em>1</em>26). In the <em>1</em> × <em>1</em>0^{<em>1</em><em>1</em>} and 5 × <em>1</em>0^<em>1</em>0 viral particles dose groups, the RBD-specific ELISA antibodies peaked at 656·5 (95% CI 575·2-749·2) and 57<em>1</em>·0 (467·6-697·3), with seroconversion rates at 96% (95% CI 93-98) and 97% (92-99), respectively, at day 28. Both doses of the vaccine induced significant neutralising antibody responses to live SARS-CoV-2, with GMTs of <em>1</em>9·5 (95% CI <em>1</em>6·8-22·7) and <em>1</em>8·3 (<em>1</em>4·4-23·3) in participants receiving <em>1</em> × <em>1</em>0^{<em>1</em><em>1</em>} and 5 × <em>1</em>0^<em>1</em>0 viral particles, respectively. Specific interferon γ enzyme-linked immunospot assay responses post vaccination were observed in 227 (90%, 95% CI 85-93) of 253 and <em>1</em><em>1</em>3 (88%, 8<em>1</em>-92) of <em>1</em>29 participants in the <em>1</em> × <em>1</em>0^{<em>1</em><em>1</em>} and 5 × <em>1</em>0^<em>1</em>0 viral particles dose groups, respectively. Solicited adverse reactions were reported by <em>1</em>83 (72%) of 253 and 96 (74%) of <em>1</em>29 participants in the <em>1</em> × <em>1</em>0^{<em>1</em><em>1</em>} and 5 × <em>1</em>0^<em>1</em>0 viral particles dose groups, respectively. Severe adverse reactions were reported by 24 (9%) participants in the <em>1</em> × <em>1</em>0^{<em>1</em><em>1</em>} viral particles dose group and one (<em>1</em>%) participant in the 5 × <em>1</em>0^<em>1</em>0 viral particles dose group. No serious adverse reactions were documented.

<strong class="sub-title"> Interpretation: </strong> The Ad5-vectored COVID-<em>1</em>9 vaccine at 5 × <em>1</em>0^<em>1</em>0 viral particles is safe, and induced significant immune responses in the majority of recipients after a single immunisation.

Funding: National Key R&D Programme of China, National Science and Technology Major Project, and CanSino Biologics.

Collection of exhaled breath condensate (EBC) is a noninvasive method for obtaining samples from the lungs. EBC contains large number of mediators including adenosine, ammonia, hydrogen peroxide, isoprostanes, leukotrienes, nitrogen oxides, peptides and cytokines. Concentrations of these mediators are influenced by lung diseases and modulated by therapeutic interventions. Similarly EBC pH also changes in respiratory diseases. The aim of the American Thoracic Society/European Respiratory Society Task Force on EBC was to identify the important methodological issues surrounding EBC collection and assay, to provide recommendations for the measurements and to highlight areas where further research is required. Based on the currently available evidence and the consensus of the expert panel for EBC collection, the following general recommendations were put together for oral sample collection: collect during tidal breathing using a noseclip and a saliva trap; define cooling temperature and collection time (<em>1</em>0 min is generally sufficient to obtain <em>1</em>-2 <em>mL</em> of sample and well tolerated by patients); use inert material for condenser; do not use resistor and do not use filter between the subject and the condenser. These are only general recommendations and certain circumstances may dictate variation from them. Important areas for future research involve: ascertaining mechanisms and site of exhaled breath condensate particle formation; determination of dilution markers; improving reproducibility; employment of EBC in longitudinal studies; and determining the utility of exhaled breath condensate measures for the management of individual patients. These studies are required before recommending this technique for use in clinical practice.

Brain expression of CB2 cannabinoid receptors has been much less well established and characterized in comparison to the expression of brain CB<em>1</em> receptors. Since CB2 receptors are intensely expressed in peripheral and immune tissues, expression in brain microglia has been anticipated. Nevertheless, we now describe expression of CB2-receptor-like immunoreactivity in brain in neuronal patterns that support broader CNS roles for this receptor. Two anti-CB2 affinity purified polyclonal antibodies were raised in rabbits immunized with peptide conjugates that corresponded to amino acids <em>1</em>-33 and 20-33. Western blot analyses revealed specific bands that were identified using these sera and were absent when the sera were preadsorbed with 8.3 mug/<em>ml</em> of the immunizing peptides. These studies, and initial RT-PCR analyses of brain CB<em>1</em> and CB2 mRNAs, also support the expression of brain CB2 receptor transcripts at levels much lower than those of CB<em>1</em> receptors. CB2 cannabinoid receptor mRNA was clearly expressed in the cerebellum of wild type but not in CB2 knockout mice. CB2 immunostaining was detected in the interpolar part of spinal 5th nucleus of wild type but not in CB2 knockout mice, using a mouse C-terminal CB2 receptor antibody. Immunohistochemical analyses revealed abundant immunostaining for CB2 receptors in apparent neuronal and glial processes in a number of rat brain areas. Cerebellar Purkinje cells and hippocampal pyramidal cells revealed substantial immunoreactivity that was absent when sections were stained with preadsorbed sera. CB2 immunoreactivity was also observed in olfactory tubercle, islands of Calleja, cerebral cortex, striatum, thalamic nuclei, hippocampus, amygdala, substantia nigra, periaqueductal gray, paratrochlear nucleus, paralemniscal nucleus, red nucleus, pontine nuclei, inferior colliculus and the parvocellular portion of the medial vestibular nucleus. In-vitro, CB2 immunoreactivity was also present in hippocampal cell cultures. The multifocal expression of CB2 immunoreactivity in glial and neuronal patterns in a number of brain regions suggests reevaluation of the possible roles that CB2 receptors may play in the brain.

OBJECTIVE

To determine the effects of longer term modest salt reduction on blood pressure, hormones, and lipids.

METHODS

Systematic review and meta-analysis.

METHODS

Medline, Embase, Cochrane Hypertension Group Specialised Register, Cochrane Central Register of Controlled Trials, and reference list of relevant articles.

METHODS

Randomised trials with a modest reduction in salt intake and duration of at least four weeks.

METHODS

Data were extracted independently by two reviewers. Random effects meta-analyses, subgroup analyses, and meta-regression were performed.

RESULTS

Thirty four trials (3230 participants) were included. Meta-analysis showed that the mean change in urinary sodium (reduced salt v usual salt) was -75 mmol/24 h (equivalent to a reduction of 4.4 g/day salt), and with this reduction in salt intake, the mean change in blood pressure was -4.<em>1</em>8 mm Hg (95% confidence interval -5.<em>1</em>8 to -3.<em>1</em>8, I(2)=75%) for systolic blood pressure and -2.06 mm Hg (-2.67 to -<em>1</em>.45, I(2)=68%) for diastolic blood pressure. Meta-regression showed that age, ethnic group, blood pressure status (hypertensive or normotensive), and the change in 24 hour urinary sodium were all significantly associated with the fall in systolic blood pressure, explaining 68% of the variance between studies. A <em>1</em>00 mmol reduction in 24 hour urinary sodium (6 g/day salt) was associated with a fall in systolic blood pressure of 5.8 mm Hg (2.5 to 9.2, P=0.00<em>1</em>) after adjustment for age, ethnic group, and blood pressure status. For diastolic blood pressure, age, ethnic group, blood pressure status, and the change in 24 hour urinary sodium explained 4<em>1</em>% of the variance between studies. Meta-analysis by subgroup showed that in people with hypertension the mean effect was -5.39 mm Hg (-6.62 to -4.<em>1</em>5, I(2)=6<em>1</em>%) for systolic blood pressure and -2.82 mm Hg (-3.54 to -2.<em>1</em><em>1</em>, I(2)=52%) for diastolic blood pressure. In normotensive people, the figures were -2.42 mm Hg (-3.56 to -<em>1</em>.29, I(2)=66%) and -<em>1</em>.00 mm Hg (-<em>1</em>.85 to -0.<em>1</em>5, I(2)=66%), respectively. Further subgroup analysis showed that the decrease in systolic blood pressure was significant in both white and black people and in men and women. Meta-analysis of data on hormones and lipids showed that the mean change was 0.26 ng/<em>mL</em>/h (0.<em>1</em>7 to 0.36, I(2)=70%) for plasma renin activity, 73.20 pmol/L (44.92 to <em>1</em>0<em>1</em>.48, I(2)=62%) for aldosterone, <em>1</em>87 pmol/L (39 to 336, I(2)=5%) for noradrenaline (norepinephrine), 37 pmol/L (-<em>1</em> to 74, I(2)=<em>1</em>2%) for adrenaline (epinephrine), 0.05 mmol/L (-0.02 to 0.<em>1</em><em>1</em>, I(2)=0%) for total cholesterol, 0.05 mmol/L (-0.0<em>1</em> to 0.<em>1</em>2, I(2)=0%) for low density lipoprotein cholesterol, -0.02 mmol/L (-0.06 to 0.0<em>1</em>, I(2)=<em>1</em>6%) for high density lipoprotein cholesterol, and 0.04 mmol/L (-0.02 to 0.09, I(2)=0%) for triglycerides.

CONCLUSIONS

A modest reduction in salt intake for four or more weeks causes significant and, from a population viewpoint, important falls in blood pressure in both hypertensive and normotensive individuals, irrespective of sex and ethnic group. Salt reduction is associated with a small physiological increase in plasma renin activity, aldosterone, and noradrenaline and no significant change in lipid concentrations. These results support a reduction in population salt intake, which will lower population blood pressure and thereby reduce cardiovascular disease. The observed significant association between the reduction in 24 hour urinary sodium and the fall in systolic blood pressure, indicates that larger reductions in salt intake will lead to larger falls in systolic blood pressure. The current recommendations to reduce salt intake from 9-<em>1</em>2 to 5-6 g/day will have a major effect on blood pressure, but a further reduction to 3 g/day will have a greater effect and should become the long term target for population salt intake.

The purpose of the present study was to test the hypothesis that a transient increase in plasma IL-6 induces an anti-inflammatory environment in humans. Therefore, young healthy volunteers received a low dose of recombinant human (rh)IL-6 or saline for 3 h. Plasma IL-6 levels during rhIL-6 infusion were approximately <em>1</em>40 pg/<em>ml</em>, corresponding to the levels obtained during strenuous exercise. The infusion of rhIL-6 did not induce enhanced levels of the proinflammatory cytokine TNF-alpha but enhanced the plasma levels of the two anti-inflammatory cytokines IL-<em>1</em> receptor agonist (IL-<em>1</em>ra) and IL-<em>1</em>0 compared with saline infusion. In addition, C-reactive protein increased 3 h post-rhIL-6 infusion and was further elevated <em>1</em>6 h later compared with saline infusion. rhIL-6 induced increased levels of plasma cortisol and, consequently, an increase in circulating neutrophils and a decrease in the lymphocyte number without effects on plasma epinephrine, body temperature, mean arterial pressure, or heart rate. In conclusion, this study demonstrates that physiological concentrations of IL-6 induce an anti-inflammatory rather than an inflammatory response in humans and that IL-6, independently of TNF-alpha, enhances the levels not only of IL-<em>1</em>ra but also of IL-<em>1</em>0. Furthermore, IL-6 induces an increase in cortisol and, consequently, in neutrocytosis and late lymphopenia to the same magnitude and with the same kinetics as during exercise, suggesting that muscle-derived IL-6 has a central role in exercise-induced leukocyte trafficking.

HDL levels are inversely related to the risk of developing atherosclerosis. In serum, paraoxonase (PON) is associated with HDL, and was shown to inhibit LDL oxidation. Whether PON also protects HDL from oxidation is unknown, and was determined in the present study. In humans, we found serum HDL PON activity and HDL susceptibility to oxidation to be inversely correlated (r2 = 0.77, n = <em>1</em>5). Supplementing human HDL with purified PON inhibited copper-induced HDL oxidation in a concentration-dependent manner. Adding PON to HDL prolonged the oxidation lag phase and reduced HDL peroxide and aldehyde formation by up to 95%. This inhibitory effect was most pronounced when PON was added before oxidation initiation. When purified PON was added to whole serum, essentially all of it became HDL-associated. The PON-enriched HDL was more resistant to copper ion-induced oxidation than was control HDL. Compared with control HDL, HDL from PON-treated serum showed a 66% prolongation in the lag phase of its oxidation, and up to a 40% reduction in peroxide and aldehyde content. In contrast, in the presence of various PON inhibitors, HDL oxidation induced by either copper ions or by a free radical generating system was markedly enhanced. As PON inhibited HDL oxidation, two major functions of HDL were assessed: macrophage cholesterol efflux, and LDL protection from oxidation. Compared with oxidized untreated HDL, oxidized PON-treated HDL caused a 45% increase in cellular cholesterol efflux from J-774 A.<em>1</em> macrophages. Both HDL-associated PON and purified PON were potent inhibitors of LDL oxidation. Searching for a possible mechanism for PON-induced inhibition of HDL oxidation revealed PON (2 paraoxonase U/<em>ml</em>)-mediated hydrolysis of lipid peroxides (by <em>1</em>9%) and of cholesteryl linoleate hydroperoxides (by 90%) in oxidized HDL. HDL-associated PON, as well as purified PON, were also able to substantially hydrolyze (up to 25%) hydrogen peroxide (H2O2), a major reactive oxygen species produced under oxidative stress during atherogenesis. Finally, we analyzed serum PON activity in the atherosclerotic apolipoprotein E-deficient mice during aging and development of atherosclerotic lesions. With age, serum lipid peroxidation and lesion size increased, whereas serum PON activity decreased. We thus conclude that HDL-associated PON possesses peroxidase-like activity that can contribute to the protective effect of PON against lipoprotein oxidation. The presence of PON in HDL may thus be a major contributor to the antiatherogenicity of this lipoprotein.

OBJECTIVE

To evaluate the association between interleukin-6, interleukin-8, and interleukin-10 and clinical outcomes including mortality in patients with acute lung injury and to determine whether lower tidal volume ventilation was associated with a decrease in plasma cytokines in patients with acute lung injury.

METHODS

Multiple-center, randomized trial.

METHODS

Intensive care units in ten university centers.

METHODS

The study included 861 patients enrolled in the National Heart, Lung and Blood Institute Acute Respiratory Distress Syndrome Clinical Network trial of lower tidal volumes compared with traditional tidal volumes for acute lung injury.

METHODS

Patients were randomized to a 6 mL/kg or a 12 mL/kg tidal volume strategy that has been previously described.

RESULTS

Baseline plasma levels of interleukin-6, interleukin-8, and interleukin-10 were each associated with an increased risk of death in both logistic regression analyses controlling for ventilator group (odds ratio 1.63 per log-10 increment, 95% confidence interval 1.33-1.98; odds ratio 2.33 per log-10 increment, 95% confidence interval 1.79-3.03; odds ratio 2.02 per log-10 increment, 95% confidence interval 1.47-2.76, respectively) and multivariate analyses controlling for ventilation strategy, Acute Physiology and Chronic Health Evaluation III score, Pao2/Fio2 ratio, creatinine, platelet count, and vasopressor use (odds ratio 1.63 per log-10 increment, 95% confidence interval 0.93-1.49; odds ratio 1.73 per log-10 increment, 95% confidence interval 1.29-2.34; odds ratio 1.23 per log-10 increment, 95% confidence interval 0.86-1.76, respectively). Interleukin-6 and interleukin-8 levels were also associated with a significant decrease in ventilator free and organ failure free days. Patients with sepsis had the highest cytokine levels and the greatest risk of death per cytokine elevation. By day 3, the 6 mL/kg strategy was associated with a greater decrease in interleukin-6 and interleukin-8 levels. There was a 26% reduction in interleukin-6 (95% confidence interval, 12-37%) and a 12% reduction in interleukin-8 (95% confidence interval, 1-23%) in the 6 mL/kg group compared with the 12 mL/kg group.

CONCLUSIONS

In patients with acute lung injury, plasma interleukin-6 and interleukin-8 levels are associated with morbidity and mortality. The severity of inflammation varies with clinical risk factor, suggesting that clinical risk factor should be considered when both developing and testing therapeutic interventions. Low tidal volume ventilation is associated with a more rapid attenuation of the inflammatory response.

Antiretroviral therapy can reduce human immunodeficiency virus type <em>1</em> (HIV-<em>1</em>) viremia to below the detection limit of ultrasensitive clinical assays (50 copies of HIV-<em>1</em> RNA/<em>ml</em>). However, latent HIV-<em>1</em> persists in resting CD4+ T cells, and low residual levels of free virus are found in the plasma. Limited characterization of this residual viremia has been done because of the low number of virions per sample. Using intensive sampling, we analyzed residual viremia and compared these viruses to latent proviruses in resting CD4+ T cells in peripheral blood. For each patient, we found some viruses in the plasma that were identical to viruses in resting CD4+ T cells by pol gene sequencing. However, in a majority of patients, the most common viruses in the plasma were rarely found in resting CD4+ T cells even when the resting cell compartment was analyzed with assays that detect replication-competent viruses. Despite the large diversity of pol sequences in resting CD4+ T cells, the residual viremia was dominated by a homogeneous population of viruses with identical pol sequences. In the most extensively studied case, a predominant plasma sequence was also found in analysis of the env gene, and linkage by long-distance reverse transcriptase PCR established that these predominant plasma sequences represented a single predominant plasma virus clone. The predominant plasma clones were released for months to years without evident sequence change. Thus, in some patients on antiretroviral therapy, the major mechanism for residual viremia involves prolonged production of a small number of viral clones without evident evolution, possibly by cells other than circulating CD4+ T cells. The sequences have been deposited in GenBank. The accession numbers are DQ 39<em>1</em>282 to DQ 39<em>1</em>35<em>1</em> (for env) and DQ 39<em>1</em>352 to DQ 392955 (for RT).

BACKGROUND

The recording of outcomes from large-scale, simplified HAART (highly active antiretroviral therapy) programmes in sub-Saharan Africa is critical. We aimed to assess the effectiveness of such a programme held by Médecins Sans Frontières (MSF) in the Chiradzulu district, Malawi.

METHODS

We scaled up and simplified HAART in this programme since August, 2002. We analysed survival indicators, CD4 count evolution, virological response, and adherence to treatment. We included adults who all started HAART 6 months or more before the analysis. HIV-<em>1</em> RNA plasma viral load and self-reported adherence were assessed on a subsample of patients, and antiretroviral resistance mutations were analysed in plasma with viral loads greater than <em>1</em>000 copies per <em>mL</em>. Analysis was by intention to treat.

RESULTS

Of the <em>1</em>308 patients who were eligible, 827 (64%) were female, the median age was 34.9 years (IQR 29.9-4<em>1</em>.0), and <em>1</em>023 (78%) received d4T/3TC/NVP (stavudine, lamivudine, and nevirapine) as a fixed-dose combination. At baseline, <em>1</em>266 individuals (97%) were HAART-naive, 357 (27%) were at WHO stage IV, 3<em>1</em><em>1</em> (33%) had a body-mass index of less than <em>1</em>8.5 kg/m2, and 208 (2<em>1</em>%) had a CD4 count lower than 50 cells per muL. At follow-up (median 8.3 months, IQR 5.5-<em>1</em>3.<em>1</em>), 967 (74%) were still on HAART, 243 (<em>1</em>9%) had died, 9<em>1</em> (7%) were lost to follow-up, and seven (0.5%) discontinued treatment. Low body-mass index, WHO stage IV, male sex, and baseline CD4 count lower than 50 cells per muL were independent determinants of death in the first 6 months. At <em>1</em>2 months, the probability of individuals still in care was 0.76 (95% CI 0.73-0.78) and the median CD4 gain was <em>1</em>65 (IQR 67-259) cells per muL. In the cross-sectional survey (n=398), 334 (84%) had a viral load of less than 400 copies per <em>mL</em>. Of several indicators measuring adherence, self-reported poor adherence (<80%) in the past 4 days was the best predictor of detectable viral load (odds ratio 5.4, 95% CI <em>1</em>.9-<em>1</em>5.6).

CONCLUSIONS

These data show that large numbers of people can rapidly benefit from antiretroviral therapy in rural resource-poor settings and strongly supports the implementation of such large-scale simplified programmes in Africa.

BACKGROUND

The primary objective of this mechanistic open-label, stratified clinical trial was to determine the effect of 8 weeks' sodium glucose cotransporter 2 inhibition with empagliflozin 25 mg QD on renal hyperfiltration in subjects with type <em>1</em> diabetes mellitus (T<em>1</em>D).

RESULTS

Inulin (glomerular filtration rate; GFR) and paraaminohippurate (effective renal plasma flow) clearances were measured in individuals stratified based on having hyperfiltration (T<em>1</em>D-H, GFR ≥ <em>1</em>35 mL/min/<em>1</em>.73m(2), n=27) or normal GFR (T<em>1</em>D-N, GFR 90-<em>1</em>34 mL/min/<em>1</em>.73m(2), n=<em>1</em>3) at baseline. Renal function and circulating levels of renin-angiotensin-aldosterone system mediators and NO were measured under clamped euglycemic (4-6 mmol/L) and hyperglycemic (9-<em>1</em><em>1</em> mmol/L) conditions at baseline and end of treatment. During clamped euglycemia, hyperfiltration was attenuated by -33 mL/min/<em>1</em>.73m(2) with empagliflozin in T<em>1</em>D-H, (GFR <em>1</em>72±23-<em>1</em>39±25 mL/min/<em>1</em>.73 m(2), P<0.0<em>1</em>). This effect was accompanied by declines in plasma NO and effective renal plasma flow and an increase in renal vascular resistance (all P<0.0<em>1</em>). Similar significant effects on GFR and renal function parameters were observed during clamped hyperglycemia. In T<em>1</em>D-N, GFR, other renal function parameters, and plasma NO were not altered by empagliflozin. Empagliflozin reduced hemoglobin A<em>1</em>c significantly in both groups, despite lower insulin doses in each group (P≤0.04).

CONCLUSIONS

In conclusion, short-term treatment with the sodium glucose cotransporter 2 inhibitor empagliflozin attenuated renal hyperfiltration in subjects with T<em>1</em>D, likely by affecting tubular-glomerular feedback mechanisms.

BACKGROUND

http://www.clinicaltrials.gov. Unique identifier: NCT0<em>1</em>392560.

OBJECTIVE

To derive a regression equation that estimates metabolic equivalent (MET) from accelerometer counts, and to define thresholds of accelerometer counts that can be used to delineate sedentary, light, moderate, and vigorous activity in adolescent girls.

METHODS

Seventy-four healthy 8th grade girls, age <em>1</em>3 - <em>1</em>4 yr, were recruited from urban areas of Baltimore, MD, Minneapolis/St. Paul, MN, and Columbia, SC, to participate in the study. Accelerometer and oxygen consumption (.-)VO(2)) data for <em>1</em>0 activities that varied in intensity from sedentary (e.g., TV watching) to vigorous (e.g., running) were collected. While performing these activities, the girls wore two accelerometers, a heart rate monitor and a Cosmed K4b2 portable metabolic unit for measurement of (.-)VO(2). A random-coefficients model was used to estimate the relationship between accelerometer counts and (.-)VO(2). Activity thresholds were defined by minimizing the false positive and false negative classifications.

RESULTS

The activities provided a wide range in (.-)VO(2) (3 - 36 <em>mL</em> x kg x min) with a correspondingly wide range in accelerometer counts (<em>1</em>- 3928 counts x 30 s). The regression line for MET score versus counts was MET = 2.0<em>1</em> +/- 0.00<em>1</em>7<em>1</em> (counts x 30 s) (mixed model R = 0.84, SEE = <em>1</em>.36). A threshold of <em>1</em>500 counts x 30 s defined the lower end of the moderate intensity (approximately 4.6 METs) range of physical activity. That cutpoint distinguished between slow and brisk walking, and gave the lowest number of false positive and false negative classifications. The threshold ranges for sedentary, light, moderate, and vigorous physical activity were found to be 0 - 50, 5<em>1</em>- <em>1</em>499, <em>1</em>500 - 2600, and >2600 counts x 30 s, respectively.

CONCLUSIONS

The developed equation and these activity thresholds can be used for prediction of MET score from accelerometer counts and participation in various intensities of physical activity in adolescent girls.

Bisphenol A (BPA), an endocrine disruptor, is employed in the manufacture of a wide range of consumer products. The suggestion that BPA, at amounts to which we are exposed, alters the reproductive organs of developing rodents has caused concern. At present, no information exists concerning the exposure of human pregnant women and their fetuses to BPA. We therefore investigated blood samples from mothers (n = 37) between weeks 32 and 4<em>1</em> of gestation. Afer the births, we also analyzed placental tissue and umbilical cord blood from the same subjects. We developed a novel chemical derivatization-gas chromatography/mass spectrometry method to analyze parent BPA at concentrations < <em>1</em> micro g/<em>mL</em> in plasma and tissues. Concentrations of BPA ranged from 0.3 to <em>1</em>8.9 ng/<em>mL</em> (median = 3.<em>1</em> ng/<em>mL</em>) in maternal plasma, from 0.2 to 9.2 ng/<em>mL</em> (median = 2.3 ng/<em>mL</em>) in fetal plasma, and from <em>1</em>.0 to <em>1</em>04.9 ng/g (median = <em>1</em>2.7 ng/g) in placental tissue. BPA blood concentrations were higher in male than in female fetuses. Here we demonstrate parent BPA in pregnant women and their fetuses. Exposure levels of parent BPA were found within a range typical of those used in recent animal studies and were shown to be toxic to reproductive organs of male and female offspring. We suggest that the range of BPA concentrations we measured may be related to sex differences in metabolization of parent BPA or variable maternal use of consumer products leaching BPA.

Vitamins D(2) and D(3) are generally considered to be equivalent in humans. Nevertheless, physicians commonly report equivocal responses to seemingly large doses of the only high-dose calciferol (vitamin D(2)) available in the U.S. market. The relative potencies of vitamins D(2) and D(3) were evaluated by administering single doses of 50,000 IU of the respective calciferols to 20 healthy male volunteers, following the time course of serum vitamin D and 25-hydroxyvitamin D (25OHD) over a period of 28 d and measuring the area under the curve of the rise in 25OHD above baseline. The two calciferols produced similar rises in serum concentration of the administered vitamin, indicating equivalent absorption. Both produced similar initial rises in serum 25OHD over the first 3 d, but 25OHD continued to rise in the D(3)-treated subjects, peaking at <em>1</em>4 d, whereas serum 25OHD fell rapidly in the D(2)-treated subjects and was not different from baseline at <em>1</em>4 d. Area under the curve (AUC) to d 28 was 60.2 ng.d/<em>ml</em> (<em>1</em>50.5 nmol.d/liter) for vitamin D(2) and 204.7 (5<em>1</em><em>1</em>.8) for vitamin D(3) (P < 0.002). Calculated AUC(infinity) indicated an even greater differential, with the relative potencies for D(3):D(2) being 9.5:<em>1</em>. Vitamin D(2) potency is less than one third that of vitamin D(3). Physicians resorting to use of vitamin D(2) should be aware of its markedly lower potency and shorter duration of action relative to vitamin D(3).

OBJECTIVE

Treatment options for metastatic melanoma are limited. We conducted this phase II trial to assess the efficacy of JS1/34.5-/47-/granulocyte-macrophage colony-stimulating factor (GM-CSF) in stages IIIc and IV disease.

METHODS

Treatment involved intratumoral injection of up to 4 mL of 10(6) pfu/mL of JS1/34.5-/47-/GM-CSF followed 3 weeks later by up to 4 mL of 10(8) pfu/mL every 2 weeks for up to 24 treatments. Clinical activity (by RECIST [Response Evaluation Criteria in Solid Tumors]), survival, and safety parameters were monitored.

RESULTS

Fifty patients (stages IIIc, n = 10; IVM1a, n = 16; IVM1b, n = 4; IVM1c, n = 20) received a median of six injection sets; 74% of patients had received one or more nonsurgical prior therapies for active disease, including dacarbazine/temozolomide or interleukin-2 (IL-2). Adverse effects were limited primarily to transient flu-like symptoms. The overall response rate by RECIST was 26% (complete response [CR], n = 8; partial response [PR], n = 5), and regression of both injected and distant (including visceral) lesions occurred. Ninety-two percent of the responses had been maintained for 7 to 31 months. Ten additional patients had stable disease (SD) for greater than 3 months, and two additional patients had surgical CR. On an extension protocol, two patients subsequently achieved CR by 24 months (one previously PR, one previously SD), and one achieved surgical CR (previously PR). Overall survival was 58% at 1 year and 52% at 24 months.

CONCLUSIONS

The 26% response rate, with durability in both injected and uninjected lesions including visceral sites, together with the survival rates, are evidence of systemic effectiveness. This effectiveness, combined with a limited toxicity profile, warrants additional evaluation of JS1/34.5-/47-/GM-CSF in metastatic melanoma. A US Food and Drug Administration-approved phase III investigation is underway.

BACKGROUND

Recent studies have shown that stem cell therapy can promote tissue regeneration; however, monitoring stem cells in vivo remains problematic owing to limitations of conventional histological assays and imaging modalities.

RESULTS

Murine embryonic stem (ES) cells were stably transduced with a lentiviral vector carrying a novel triple-fusion (TF) reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). ES cell viability, proliferation, and differentiation ability were not adversely affected by either reporter genes or reporter probes compared with nontransduced control cells (P=NS). Afterward, <em>1</em>x<em>1</em>0(7) of ES cells carrying the TF reporter gene (ES-TF) were injected into the myocardium of adult nude rats (n=20). Control animals received nontransduced ES cells (n=6). At day 4, the bioluminescence and positron emission tomography signals in study animals were 3.7x<em>1</em>0(7)+/-5.8x<em>1</em>0(6) photons.s(-<em>1</em>).cm(-2) per steradian (sr) and 0.08+/-0.03% injected dose/g, respectively (P<0.05 versus control). Both signals increased progressively from week <em>1</em> to week 4, which indicated ES cell survival and proliferation in the host. Histological analysis demonstrated the formation of intracardiac and extracardiac teratomas. Finally, animals (n=4) that were treated with intraperitoneal injection of ganciclovir (50 mg/kg) did not develop teratomas when compared with control animals (n=4) treated with saline (<em>1</em> <em>mL</em>/kg).

CONCLUSIONS

This is the first study to characterize ES cells that stably express fluorescence, bioluminescence, and positron emission tomography reporter genes and monitor the kinetics of ES cell survival, proliferation, and migration. This versatile imaging platform should have broad applications for basic research and clinical studies on stem cell therapy.

<em>1</em>. End-plate currents have been studied in glycerol-treated frog sartorius nerve-muscle preparations with the voltage clamp technique.2. End-plate currents follow a simple exponential time course over most of their declining phase.3. The rate constant alpha that characterizes this exponential decay depends upon membrane potential V according to the relationship alpha (V) = Be(AV), with A = 0.00795 +/- 0.00043 (S.E.) mV(-<em>1</em>) and B = <em>1</em>.67 +/- 0.04 (S.E.) msec(-<em>1</em>).4. Voltage sensitivity decreases (that is, A in the above equation becomes smaller) as the recording and current-passing electrodes are moved away from the end-plate region.5. The voltage sensitivity of alpha is decreased by decreasing the gain of the voltage clamp amplifier.6. Changing the end-plate current amplitude by curare treatment, by increased calcium ion concentration, and by facilitation and depression has essentially no effect on end-plate current time course.7. When membrane potential is changed step-wise during the decaying phase of the end-plate conductance change, currents begin to decline with a rate constant alpha appropriate to the new membrane potential in less than 0.2 msec.8. Treatment with prostigmine methylsulphate in concentrations up to 50 mug/<em>ml</em>. slows end-plate current decay but has little effect on voltage sensitivity. That is, B in the above equation is decreased by prostigmine treatment, but A is relatively unaffected.

Most animal studies using passive administration of HIV broadly neutralizing monoclonal antibodies (bnMAbs) have associated protection against high-dose mucosal viral challenge with relatively high serum concentrations of antibody. We recently identified several bnMAbs remarkable for their in vitro potency against HIV. Of these bnMAbs, PGT<em>1</em>2<em>1</em> is one of the most broad and potent antibodies isolated to date and shows <em>1</em>0- to <em>1</em>00-fold higher neutralizing activity than previously characterized bnMAbs. To evaluate the protective potency of PGT<em>1</em>2<em>1</em> in vivo, we performed a protection study in rhesus macaques. Animals were i.v. administered 5 mg/kg, <em>1</em> mg/kg, or 0.2 mg/kg PGT<em>1</em>2<em>1</em> 24 h before being vaginally challenged with a single high dose of chimeric simian-human immunodeficiency virus (SHIV)(SF<em>1</em>62P3). Sterilizing immunity was achieved in all animals administered 5 mg/kg and <em>1</em> mg/kg and three of five animals administered 0.2 mg/kg PGT<em>1</em>2<em>1</em>, with corresponding average antibody serum concentrations of 95 µg/<em>mL</em>, <em>1</em>5 µg/<em>mL</em>, and <em>1</em>.8 µg/<em>mL</em>, respectively. The results suggest that a protective serum concentration for PGT<em>1</em>2<em>1</em> is in the single-digit µg/<em>mL</em> for SHIV(SF<em>1</em>62P3), showing that PGT<em>1</em>2<em>1</em> can mediate sterilizing immunity at serum concentrations that are significantly lower than those observed in previous studies and that may be achievable through vaccination with the development of a suitable immunogen.