BACKGROUND

B-type or brain natriuretic peptide (BNP) is a novel natriuretic peptide secreted from the heart that forms a peptide family with A-type or atrial natriuretic peptide (ANP), and its plasma level has been shown to be increased in patients with congestive heart failure. This study was designed to examine the sources and mechanisms of the secretion of BNP in comparison with those of ANP in control subjects and in patients with heart failure.

RESULTS

We measured the plasma levels of BNP as well as ANP in 16 patients with dilated cardiomyopathy (11 men and 5 women; mean age, 59 years) and 18 control subjects (9 men and 9 women; mean age, 54 years) by sampling blood from the femoral vein, the aortic root, the anterior interventricular vein (AIV), and the coronary sinus using the newly developed immunoradiometric assay systems. In the control subjects, there was no significant difference in the plasma ANP level between the aortic root and the AIV (24.0 +/- 5.2 pg/mL versus 32.2 +/- 17.0 pg/mL), but there was a highly significant step-up of the level between the AIV and the coronary sinus (32.2 +/- 17.0 pg/mL versus 371.4 +/- 111.1 pg/mL, P < .001). In contrast, there was a significant step-up of the plasma BNP level between the aortic root and the AIV (8.6 +/- 6.4 pg/mL versus 19.0 +/- 11.5 pg/mL, P < .01) but not between the AIV and the coronary sinus (19.0 +/- 11.5 pg/mL versus 28.8 +/- 14.0 pg/mL). On the other hand, in patients with dilated cardiomyopathy, there was a significant step-up in the plasma ANP level between the aortic root and the AIV (280.6 +/- 183.7 pg/mL versus 612.3 +/- 431.6 pg/mL, P < .01) and between the AIV and the coronary sinus (612.3 +/- 431.6 pg/mL versus 1229.0 +/- 772.7 pg/mL, P < .01). There was a significant step-up in the plasma BNP level between the aortic root and the AIV (268.4 +/- 293.2 pg/mL versus 511.6 +/- 458.1 pg/mL, P < .01) but not between the AIV and the coronary sinus (511.6 +/- 458.1 pg/mL versus 529.7 +/- 455.3 pg/mL) in patients with dilated cardiomyopathy. The arteriovenous difference at the AIV of the plasma level of BNP had a significant positive correlation with left ventricular end-systolic volume index (r = 0.859, P < .001) and a significant negative correlation with left ventricular ejection fraction (r = -.735, P < .001).

CONCLUSIONS

We conclude that (1) BNP is secreted mainly from the left ventricle in normal adult humans as well as in patients with left ventricular dysfunction, whereas ANP is secreted from atria in normal adult humans and also from the left ventricle in patients with left ventricular dysfunction; (2) secretion of BNP as well as ANP from the left ventricle increases in proportion to the severity of the left ventricular dysfunction, suggesting that the secretions of ANP and BNP from the left ventricle are regulated mainly by wall tension of the left ventricle; and (3) the peripheral plasma levels of ANP and BNP reflect the secretion rate of these hormones from the left ventricle and may be used as a marker of the degree of left ventricular dysfunction in patients with left ventricular dysfunction.

The aim of this study was to investigate whether interleukin (IL)-6 induces the production of IL-<em>1</em> and tumor necrosis factor (TNF) antagonists. Serial plasma samples were obtained from cancer patients participating in phase I and II trials of recombinant IL-6 administered as a <em>1</em>20-hour continuous intravenous (IV) infusion. Plasma IL-<em>1</em> receptor antagonist (IL-<em>1</em>Ra) and soluble TNF receptor p55 (TNFsRp55) levels were measured by specific radioimmunoassays (RIAs). IL-<em>1</em>Ra levels increased rapidly, reaching peak values (9.6 +/- <em>1</em>.7 ng/<em>mL</em>) within 2 to 4 hours of beginning treatment. Thereafter, levels promptly declined, reaching near baseline within 24 hours despite continuation of IL-6. TNFsRp55 plasma levels increased within 4 to 8 hours after initiating treatment and increased progressively throughout the duration of therapy. IL-<em>1</em> beta and TNF-alpha plasma levels were below the detection limit in all samples tested. Peripheral blood mononuclear cells (PBMC) exposed to IL-6 produced only small amounts (<em>1</em>.56 +/- 0.3 ng/<em>mL</em>) of IL-<em>1</em>Ra, even in the presence of exogenous soluble IL-6 receptor (gp80). TNFsRp55 levels measured in the supernatants of IL-6-stimulated PBMC were below the detection limit of the assay. Macrophages generated by culturing monocytes in granulocyte-macrophage colony-stimulating factor (GM-CSF) were much more responsive to IL-6 than freshly isolated unfractionated or adherent PBMC and synthesized almost as much IL-<em>1</em>Ra when stimulated with IL-6 as with endotoxin. These results suggest that the antinflammatory properties of IL-6 may be due; in part, to the induction of IL-<em>1</em>Ra synthesis and the release of soluble TNF receptors. Our findings also suggest that tissue macrophages may be an important source of IL-6-induced IL-<em>1</em>Ra.

OBJECTIVE

To describe trends of primary efficacy and safety outcomes of islet transplantation in type <em>1</em> diabetes recipients with severe hypoglycemia from the Collaborative Islet Transplant Registry (CITR) from <em>1</em>999 to 20<em>1</em>0.

METHODS

A total of 677 islet transplant-alone or islet-after-kidney recipients with type <em>1</em> diabetes in the CITR were analyzed for five primary efficacy outcomes and overall safety to identify any differences by early (<em>1</em>999-2002), mid (2003-2006), or recent (2007-20<em>1</em>0) transplant era based on annual follow-up to 5 years.

RESULTS

Insulin independence at 3 years after transplant improved from 27% in the early era (<em>1</em>999-2002, n = 2<em>1</em>4) to 37% in the mid (2003-2006, n = 255) and to 44% in the most recent era (2007-20<em>1</em>0, n = 208; P = 0.006 for years-by-era; P = 0.0<em>1</em> for era alone). C-peptide ≥0.3 ng/mL, indicative of islet graft function, was retained longer in the most recent era (P < 0.00<em>1</em>). Reduction of HbA(<em>1</em>c) and resolution of severe hypoglycemia exhibited enduring long-term effects. Fasting blood glucose stabilization also showed improvements in the most recent era. There were also modest reductions in the occurrence of adverse events. The islet reinfusion rate was lower: 48% by <em>1</em> year in 2007-20<em>1</em>0 vs. 60-65% in <em>1</em>999-2006 (P < 0.0<em>1</em>). Recipients that ever achieved insulin-independence experienced longer duration of islet graft function (P < 0.00<em>1</em>).

CONCLUSIONS

The CITR shows improvement in primary efficacy and safety outcomes of islet transplantation in recipients who received transplants in 2007-20<em>1</em>0 compared with those in <em>1</em>999-2006, with fewer islet infusions and adverse events per recipient.

BACKGROUND

Incidence of end-stage renal disease due to hypertension has increased in recent decades, but the optimal strategy for treatment of hypertension to prevent renal failure is unknown, especially among African Americans.

OBJECTIVE

To compare the effects of an angiotensin-converting enzyme (ACE) inhibitor (ramipril), a dihydropyridine calcium channel blocker (amlodipine), and a beta-blocker (metoprolol) on hypertensive renal disease progression.

METHODS

Interim analysis of a randomized, double-blind, 3 x 2 factorial trial conducted in 1094 African Americans aged 18 to 70 years with hypertensive renal disease (glomerular filtration rate [GFR] of 20-65 mL/min per 1.73 m(2)) enrolled between February 1995 and September 1998. This report compares the ramipril and amlodipine groups following discontinuation of the amlodipine intervention in September 2000.

METHODS

Participants were randomly assigned to receive amlodipine, 5 to 10 mg/d (n = 217), ramipril, 2.5 to 10 mg/d (n = 436), or metoprolol, 50 to 200 mg/d (n = 441), with other agents added to achieve 1 of 2 blood pressure goals.

METHODS

The primary outcome measure was the rate of change in GFR; the main secondary outcome was a composite index of the clinical end points of reduction in GFR of more than 50% or 25 mL/min per 1.73 m(2), end-stage renal disease, or death.

RESULTS

Among participants with a urinary protein to creatinine ratio of >0.22 (corresponding approximately to proteinuria of more than 300 mg/d), the ramipril group had a 36% (2.02 [SE, 0.74] mL/min per 1.73 m(2)/y) slower mean decline in GFR over 3 years (P =.006) and a 48% reduced risk of the clinical end points vs the amlodipine group (95% confidence interval [CI], 20%-66%). In the entire cohort, there was no significant difference in mean GFR decline from baseline to 3 years between treatment groups (P =.38). However, compared with the amlodipine group, after adjustment for baseline covariates the ramipril group had a 38% reduced risk of clinical end points (95% CI, 13%-56%), a 36% slower mean decline in GFR after 3 months (P =.002), and less proteinuria (P<.001).

CONCLUSIONS

Ramipril, compared with amlodipine, retards renal disease progression in patients with hypertensive renal disease and proteinuria and may offer benefit to patients without proteinuria.

The nonhematopoietic component of bone marrow includes multipotent mesenchymal stem cells (MSC) capable of differentiating into fat, bone, muscle, cartilage, and endothelium. In this report, we describe the cell culture and characterization, delivery system, and successful use of topically applied autologous MSC to accelerate the healing of human and experimental murine wounds. A single bone marrow aspirate of 35-50 <em>mL</em> was obtained from patients with acute wounds (n = 5) from skin cancer surgery and from patients with chronic, long-standing, nonhealing lower extremity wounds (n = 8). Cells were grown in vitro under conditions favoring the propagation of MSC, and flow cytometry and immunostaining showed a profile (CD29+, CD44+, CD<em>1</em>05+, CD<em>1</em>66+, CD34-, CD45-) highly consistent with published reports of human MSC. Functional induction studies confirmed that the MSC could differentiate into bone, cartilage, and adipose tissue. The cultured autologous MSC were applied up to four times to the wounds using a fibrin polymer spray system with a double-barreled syringe. Both fibrinogen (containing the MSC) and thrombin were diluted to optimally deliver a polymerized gel that immediately adhered to the wound, without run-off, and yet allowing the MSC to remain viable and migrate from the gel. Sequential adjacent sections from biopsy specimens of the wound bed after MSC application showed elongated spindle cells, similar to their in vitro counterparts, which immunostained for MSC markers. Generation of new elastic fibers was evident by both special stains and antibodies to human elastin. The application of cultured cells was safe, without treatment-related adverse events. A strong direct correlation was found between the number of cells applied (greater than <em>1</em> x <em>1</em>0(6) cells per cm2 of wound area) and the subsequent decrease in chronic wound size (p = 0.0058). Topical application of autologous MSC also stimulated closure of full-thickness wounds in diabetic mice (db/db). Tracking of green fluorescent protein (GFP)+ MSC in mouse wounds showed GFP+ blood vessels, suggesting that the applied cells may persist as well as act to stimulate the wound repair process. These findings indicate that autologous bone marrow-derived MSC can be safely and effectively delivered to wounds using a fibrin spray system.

<strong class="sub-title"> Background: </strong> With the unprecedented morbidity and mortality associated with the COVID-<em>1</em>9 pandemic, a vaccine against COVID-<em>1</em>9 is urgently needed. We investigated CoronaVac (Sinovac Life Sciences, Beijing, China), an inactivated vaccine candidate against COVID-<em>1</em>9, containing inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for its safety, tolerability and immunogenicity.

<strong class="sub-title"> Methods: </strong> In this randomised, double-blind, placebo-controlled, phase <em>1</em>/2 clinical trial, healthy adults aged <em>1</em>8-59 years were recruited from the community in Suining County of Jiangsu province, China. Adults with SARS-CoV-2 exposure or infection history, with axillary temperature above 37·0°C, or an allergic reaction to any vaccine component were excluded. The experimental vaccine for the phase <em>1</em> trial was manufactured using a cell factory process (CellSTACK Cell Culture Chamber <em>1</em>0, Corning, Wujiang, China), whereas those for the phase 2 trial were produced through a bioreactor process (ReadyToProcess WAVE 25, GE, Umea, Sweden). The phase <em>1</em> trial was done in a dose-escalating manner. At screening, participants were initially separated (<em>1</em>:<em>1</em>), with no specific randomisation, into two vaccination schedule cohorts, the days 0 and <em>1</em>4 vaccination cohort and the days 0 and 28 vaccination cohort, and within each cohort the first 36 participants were assigned to block <em>1</em> (low dose CoronaVac [3 μg per 0·5 <em>mL</em> of aluminium hydroxide diluent per dose) then another 36 were assigned to block 2 (high-dose Coronavc [6 μg per 0·5 <em>mL</em> of aluminium hydroxide diluent per dse]). Within each block, participants were randomly assigned (2:<em>1</em>), using block randomisation with a block size of six, to either two doses of CoronaVac or two doses of placebo. In the phase 2 trial, at screening, participants were initially separated (<em>1</em>:<em>1</em>), with no specific randomisation, into the days 0 and <em>1</em>4 vaccination cohort and the days 0 and 28 vaccination cohort, and participants were randomly assigned (2:2:<em>1</em>), using block randomisation with a block size of five, to receive two doses of either low-dose CoronaVac, high-dose CoronaVac, or placebo. Participants, investigators, and laboratory staff were masked to treatment allocation. The primary safety endpoint was adverse reactions within 28 days after injection in all participants who were given at least one dose of study drug (safety population). The primary immunogenic outcome was seroconversion rates of neutralising antibodies to live SARS-CoV-2 at day <em>1</em>4 after the last dose in the days 0 and <em>1</em>4 cohort, and at day 28 after the last dose in the days 0 and 28 cohort in participants who completed their allocated two-dose vaccination schedule (per-protocol population). This trial is registered with ClinicalTrials.gov, <a href="http://clinicaltrials.gov/show/NCT04352608" title="See in ClinicalTrials.gov">NCT04352608</a>, and is closed to accrual.

<strong class="sub-title"> Findings: </strong> Between April <em>1</em>6 and April 25, 2020, <em>1</em>44 participants were enrolled in the phase <em>1</em> trial, and between May 3 and May 5, 2020, 600 participants were enrolled in the phase 2 trial. 743 participants received at least one dose of investigational product (n=<em>1</em>43 for phase <em>1</em> and n=600 for phase 2; safety population). In the phase <em>1</em> trial, the incidence of adverse reactions for the days 0 and <em>1</em>4 cohort was seven (29%) of 24 participants in the 3 ug group, nine (38%) of 24 in the 6 μg group, and two (8%) of 24 in the placebo group, and for the days 0 and 28 cohort was three (<em>1</em>3%) of 24 in the 3 μg group, four (<em>1</em>7%) of 24 in the 6 μg group, and three (<em>1</em>3%) of 23 in the placebo group. The seroconversion of neutralising antibodies on day <em>1</em>4 after the days 0 and <em>1</em>4 vaccination schedule was seen in <em>1</em><em>1</em> (46%) of 24 participants in the 3 μg group, <em>1</em>2 (50%) of 24 in the 6 μg group, and none (0%) of 24 in the placebo group; whereas at day 28 after the days 0 and 28 vaccination schedule, seroconversion was seen in 20 (83%) of 24 in the 3 μg group, <em>1</em>9 (79%) of 24 in the 6 μg group, and one (4%) of 24 in the placebo group. In the phase 2 trial, the incidence of adverse reactions for the days 0 and <em>1</em>4 cohort was 40 (33%) of <em>1</em>20 participants in the 3 μg group, 42 (35%) of <em>1</em>20 in the 6 μg group, and <em>1</em>3 (22%) of 60 in the placebo group, and for the days 0 and 28 cohort was 23 (<em>1</em>9%) of <em>1</em>20 in the 3 μg group, 23 (<em>1</em>9%) of <em>1</em>20 in the 6 μg group, and <em>1</em><em>1</em> (<em>1</em>8%) of 60 for the placebo group. Seroconversion of neutralising antibodies was seen for <em>1</em>09 (92%) of <em>1</em><em>1</em>8 participants in the 3 μg group, <em>1</em><em>1</em>7 (98%) of <em>1</em><em>1</em>9 in the 6 μg group, and two (3%) of 60 in the placebo group at day <em>1</em>4 after the days 0 and <em>1</em>4 schedule; whereas at day 28 after the days 0 and 28 schedule, seroconversion was seen in <em>1</em><em>1</em>4 (97%) of <em>1</em><em>1</em>7 in the 3 μg group, <em>1</em><em>1</em>8 (<em>1</em>00%) of <em>1</em><em>1</em>8 in the 6 μg group, and none (0%) of 59 in the placebo group.

Interpretation: Taking safety, immunogenicity, and production capacity into account, the 3 μg dose of CoronaVac is the suggested dose for efficacy assessment in future phase 3 trials.

Funding: Chinese National Key Research and Development Program and Beijing Science and Technology Program.

The isolation of human monoclonal antibodies is providing important insights into the specificities that underlie broad neutralization of HIV-<em>1</em> (reviewed in ref. <em>1</em>). Here we report a broad and extremely potent HIV-specific monoclonal antibody, termed 35O22, which binds a novel HIV-<em>1</em> envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of <em>1</em>8<em>1</em> pseudoviruses with a half-maximum inhibitory concentration (IC50) <50 μg <em>ml</em>(-<em>1</em>). The median IC50 of neutralized viruses was 0.033 μg <em>ml</em>(-<em>1</em>), among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp<em>1</em>20 and gp4<em>1</em>. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design.

BACKGROUND

We examined specific causes of mortality in human immunodeficiency virus type <em>1</em> (HIV-<em>1</em>)-infected patients who initiated antiretroviral therapy (ART) in Europe and North America from <em>1</em>996 through 2006, and we quantified associations of prognostic factors with cause-specific mortality.

METHODS

We retrospectively classified all deaths among 39,272 patients enrolled in <em>1</em>3 HIV-<em>1</em> cohorts (<em>1</em>54,667 person years of follow-up) into the categories specified in the Cause of Death (CoDe) project protocol.

RESULTS

In <em>1</em>597 (85%) of <em>1</em>876 deaths, a definitive cause of death could be assigned. Among these, 792 deaths (49.5%) were AIDS related, followed by non-AIDS malignancies (<em>1</em>89; <em>1</em><em>1</em>.8%), non-AIDS infections (<em>1</em>3<em>1</em>; 8.2%), violence- and/or drug-related causes (<em>1</em>24; 7.7%), liver disease (<em>1</em><em>1</em>3; 7.0%), and cardiovascular disease (<em>1</em>03; 6.5%). Rates of AIDS-related death (hazard ratio [HR] per <em>1</em>00 cell decrease, <em>1</em>.43; 95% confidence interval [CI], <em>1</em>.34-<em>1</em>.53) and death from renal failure (HR, <em>1</em>.73; 95% CI, <em>1</em>.<em>1</em>8-2.55) were strongly inversely related to CD4 count at initiation of ART, whereas rates of death attributable to AIDS (HR for viral load >5 vs 5 log copies/mL, <em>1</em>.3<em>1</em>; 95% CI, <em>1</em>.<em>1</em>2-<em>1</em>.53), infection (HR, <em>1</em>.85; 95% CI, <em>1</em>.25-2.73), cardiovascular (HR, <em>1</em>.54; 95% CI, <em>1</em>.05-2.27), and respiratory causes (HR, 3.62; 95% CI, <em>1</em>.30-<em>1</em>0.09) were higher in patients with baseline viral load >5 log copies/mL than in other patients. Rates of each cause of death were higher in patients with presumed transmission via injection drug use than in other patients, with marked increases in rates of liver-related (HR for injection drug use vs non-injection drug use, 6.06; 95% CI, 4.03-9.09) and respiratory tract-related (HR, 4.94; 95% CI, <em>1</em>.96-<em>1</em>2.45) mortality. The proportion of deaths classified as AIDS related decreased with increasing duration of ART.

CONCLUSIONS

Important contributors to non-AIDS mortality in treated HIV-<em>1</em>-infected individuals must be addressed if decreases in mortality rates are to continue.

Marked abnormalities in cardiovascular function accompany septic shock, and bacterial endotoxin is believed to be one of the principal mediators of these abnormalities. To evaluate the cardiovascular effects of endotoxemia in humans, we measured hemodynamic variables in nine normal subjects given an intravenous bolus dose of endotoxin (Escherichia coli, 4 ng per kilogram of body weight) and in six normal subjects given a bolus dose of saline, before and three hours after administration. All the subjects then underwent volume loading with normal saline (mean, 22<em>1</em>7 <em>ml</em>) during the fourth and fifth hours after administration of the bolus, and the measurements were repeated. Three hours after the administration of endotoxin and before volume loading, the cardiac index had increased by 53 percent and the heart rate by 36 percent (both changes were significant; P less than or equal to 0.008), and the systemic vascular-resistance index had decreased by 46 percent (P = 0.004). After volume loading (five hours after the administration of endotoxin), the left ventricular ejection fraction decreased by <em>1</em> percent of the base-line value in the subjects given endotoxin, but increased by <em>1</em>4 percent in the controls (P = 0.008). The left ventricular end-diastolic and end-systolic volume indexes increased by <em>1</em>8 percent (P = 0.07) and 24 percent (P = 0.042), respectively. Left ventricular performance, as measured by the ratio of the peak systolic pressure to the end-systolic volume index, was depressed (a decrease of 0.90 in the subjects given endotoxin vs. an increase of 0.76 in the controls; P = 0.024). We conclude that the administration of endotoxin to normal subjects causes a depression of left ventricular function that is independent of changes in left ventricular volume or vascular resistance. The changes in function are similar to those observed in septic shock and suggest that endotoxin is a major mediator of the cardiovascular dysfunction in this condition.

Primary pulmonary hypertension (PPH) is characterized by the proliferation of smooth-muscle cells, fibroblasts, and endothelial cells in the walls of small pulmonary arteries. In order to evaluate a role for proinflammatory cytokines in this process, we studied the concentration of interleukin-<em>1</em> beta (IL-<em>1</em> beta), IL-6, and tumor necrosis factor-alpha (TNF alpha) in the serum of 29 patients with severe PPH referred to our center for lung transplantation. Results were compared with those obtained in <em>1</em>5 normal controls and nine patients with pulmonary hypertension secondary to chronic obstructive pulmonary disease (COPD-PH). TNF alpha serum levels were within the normal range in each group. This contrasted with increased IL-<em>1</em> beta serum levels in severe PPH (<em>1</em><em>1</em>8 +/- 36 pg/<em>ml</em>, mean +/- SEM) as compared with controls (3 +/- <em>1</em> pg/<em>ml</em>, p < 0.00<em>1</em>) or COPD-PH patients (3 +/- <em>1</em> pg/<em>ml</em>, p < 0.00<em>1</em>). IL-6 serum concentrations were also higher in severe PPH (66 +/- 20 pg/<em>ml</em>) than in controls (<em>1</em>4 +/- 6 pg/<em>ml</em>, p < 0.0<em>1</em>). This study demonstrates increased serum levels of IL-<em>1</em> beta and IL-6 in severe PPH, and suggests a role for proinflammatory cytokines in PPH.

<AbstractText>The outbreak of the coronavirus disease 20<em>1</em>9 (Covid-<em>1</em>9) shows a global spreading trend. Early and effective predictors of clinical outcomes is urgent needed to improve management of Covid-<em>1</em>9 patients.</AbstractText><AbstractText>The aim of the present study was to evaluate whether elevated D-dimer levels could predict mortality in patients with Covid-<em>1</em>9.</AbstractText><AbstractText>Patients with laboratory confirmed Covid-<em>1</em>9 were retrospective enrolled in Wuhan Asia General Hospital from January <em>1</em>2, 2020 to March <em>1</em>5, 2020. D-dimer levels on admission, and death events were collected to calculate the optimum cutoff using receiver operating characteristic curve. According to the cutoff, the subjects were divided into two groups. Then the in-hospital mortality between two groups were compared to assess the predictive value of D-dimer level.</AbstractText><AbstractText>A total of 343 eligible patients were enrolled in the study. The optimum cutoff value of D-dimer to predict in-hospital mortality was 2.0 µg/<em>ml</em> with a sensitivity of 92.3% and a specificity of 83.3%. There were 67 patients with D-dimer≥2.0 µg/<em>ml</em>, and 267 patients with D-dimer <2.0 µg/<em>ml</em> on admission. <em>1</em>3 deaths occurred during hospitalization. Patients with D-dimer levels≥2.0 µg/<em>ml</em> had a higher incidence of mortality when comparing to those who with D-dimer levels < 2.0 µg/<em>ml</em> (<em>1</em>2/67 vs <em>1</em>/267, P<0.00<em>1</em>, HR:5<em>1</em>.5, 95%CI:<em>1</em>2.9-206.7).</AbstractText><AbstractText>D-dimer on admission greater than 2.0µg/<em>mL</em> (fourfold increase) could effectively predict in-hospital mortality in patients with Covid-<em>1</em>9, which indicated D-dimer could be an early and helpful marker to improve management of Covid-<em>1</em>9 patients.</AbstractText>

A simple, fast, and inexpensive method for the determination of pesticide residues in fruits and vegetables is introduced. The procedure involves initial single-phase extraction of <em>1</em>0 g sample with <em>1</em>0 <em>mL</em> acetonitrile, followed by liquid-liquid partitioning formed by addition of 4 g anhydrous MgSO4 plus <em>1</em> g NaCl. Removal of residual water and cleanup are performed simultaneously by using a rapid procedure called dispersive solid-phase extraction (dispersive-SPE), in which <em>1</em>50 mg anhydrous MgSO4 and 25 mg primary secondary amine (PSA) sorbent are simply mixed with <em>1</em> <em>mL</em> acetonitrile extract. The dispersive-SPE with PSA effectively removes many polar matrix components, such as organic acids, certain polar pigments, and sugars, to some extent from the food extracts. Gas chromatography/mass spectrometry (GC/MS) is then used for quantitative and confirmatory analysis of GC-amenable pesticides. Recoveries between 85 and <em>1</em>0<em>1</em>% (mostly>> 95%) and repeatabilities typically < 5% have been achieved for a wide range of fortified pesticides, including very polar and basic compounds such as methamidophos, acephate, omethoate, imazalil, and thiabendazole. Using this method, a single chemist can prepare a batch of 6 previously chopped samples in < 30 min with approximately <em>1</em> dollar (U.S.) of materials per sample.

<strong class="sub-title">Background:</strong> Mortality of patients with coronavirus disease 20<em>1</em>9 (COVID-<em>1</em>9), acute respiratory distress syndrome (ARDS), and systemic inflammation is high. In areas of pandemic outbreak, the number of patients can exceed maximum capacity of intensive care units (ICUs), and, thus, these individuals often receive non-invasive ventilation outside of the ICU. Effective treatments for this population are needed urgently. Anakinra is a recombinant interleukin-<em>1</em> receptor antagonist that might be beneficial in this patient population.

<strong class="sub-title">Methods:</strong> We conducted a retrospective cohort study at the San Raffaele Hospital in Milan, Italy. We included consecutive patients (aged ≥<em>1</em>8 years) with COVID-<em>1</em>9, moderate-to-severe ARDS, and hyperinflammation (defined as serum C-reactive protein ≥<em>1</em>00 mg/L, ferritin ≥900 ng/mL, or both) who were managed with non-invasive ventilation outside of the ICU and who received standard treatment of 200 mg hydroxychloroquine twice a day orally and 400 mg lopinavir with <em>1</em>00 mg ritonavir twice a day orally. We compared survival, mechanical ventilation-free survival, changes in C-reactive protein, respiratory function, and clinical status in a cohort of patients who received additional treatment with anakinra (either 5 mg/kg twice a day intravenously [high dose] or <em>1</em>00 mg twice a day subcutaneously [low dose]) with a retrospective cohort of patients who did not receive anakinra (referred to as the standard treatment group). All outcomes were assessed at 2<em>1</em> days. This study is part of the COVID-<em>1</em>9 Biobank study, which is registered with ClinicalTrials.gov, <a href= "http://clinicaltrials.gov/show/NCT043<em>1</em>8366" title= "See in ClinicalTrials.gov">NCT043<em>1</em>8366</a>.

<strong class="sub-title">Findings:</strong> Between March <em>1</em>7 and March 27, 2020, 29 patients received high-dose intravenous anakinra, non-invasive ventilation, and standard treatment. Between March <em>1</em>0 and March <em>1</em>7, 2020, <em>1</em>6 patients received non-invasive ventilation and standard treatment only and comprised the comparison group for this study. A further seven patients received low-dose subcutaneous anakinra in addition to non-invasive ventilation and standard treatment; however, anakinra treatment was interrupted after 7 days because of a paucity of effects on serum C-reactive protein and clinical status. At 2<em>1</em> days, treatment with high-dose anakinra was associated with reductions in serum C-reactive protein and progressive improvements in respiratory function in 2<em>1</em> (72%) of 29 patients; five (<em>1</em>7%) patients were on mechanical ventilation and three (<em>1</em>0%) died. In the standard treatment group, eight (50%) of <em>1</em>6 patients showed respiratory improvement at 2<em>1</em> days; one (6%) patient was on mechanical ventilation and seven (44%) died. At 2<em>1</em> days, survival was 90% in the high-dose anakinra group and 56% in the standard treatment group (p=0·009). Mechanical ventilation-free survival was 72% in the anakinra group versus 50% in the standard treatment group (p=0·<em>1</em>5). Bacteraemia occurred in four (<em>1</em>4%) of 29 patients receiving high-dose anakinra and two (<em>1</em>3%) of <em>1</em>6 patients receiving standard treatment. Discontinuation of anakinra was not followed by inflammatory relapses.

<strong class="sub-title">Interpretation:</strong> In this retrospective cohort study of patients with COVID-<em>1</em>9 and ARDS managed with non-invasive ventilation outside of the ICU, treatment with high-dose anakinra was safe and associated with clinical improvement in 72% of patients. Confirmation of efficacy will require controlled trials.

Funding: None.

BACKGROUND

In patients with aggressive malignancies who are undergoing high-dose chemotherapy, even minimal elevation of troponin I (TnI) is associated with late left ventricular dysfunction. The time course of the subclinical myocardial damage and its impact on the clinical outcome have never been investigated previously.

RESULTS

In 703 cancer patients, we measured TnI soon after chemotherapy (early TnI) and <em>1</em> month later (late TnI). Troponin was considered positive for values>> or =0.08 ng/<em>mL</em>. Clinical and left ventricular ejection fraction evaluation (echocardiography) were performed before chemotherapy, <em>1</em>, 3, 6, and <em>1</em>2 months after the end of the treatment, and again every 6 months afterward. Three different TnI patterns were identified, and patients were grouped accordingly. In 495 patients, both early and late TnI values were <0.08 ng/<em>mL</em> (TnI-/- group); in <em>1</em>45, there was only an early increase (TnI+/- group); and in 63 patients, both values increased (TnI+/+ group). In the TnI-/- group, no significant reduction in ejection fraction was observed during the follow-up, and there was a very low incidence of cardiac events (<em>1</em>%). In contrast, a greater incidence of cardiac events occurred in TnI-positive patients, particularly in the TnI(+/+) group (84% versus 37% in the TnI+/- group; P<0.00<em>1</em>).

CONCLUSIONS

TnI release pattern after high-dose chemotherapy identifies patients at different risks of cardiac events in the 3 years thereafter. This stratification allows us to differentiate the monitoring program and to plan, in selected patients, preventive strategies aimed at improving clinical outcome.

As procalcitonin concentrations have been shown to be elevated in patients with septicemia and gram-negative infections in particular, we proceeded to investigate the effect of endotoxin, a product of gram-negative bacteria, on procalcitonin concentrations in normal human volunteers. Endotoxin from Escherichia coli 0<em>1</em><em>1</em>3:H<em>1</em>0:k, was injected i.v. at a dose of 4 mg/kg BW into these healthy volunteers. Blood samples were obtained before and <em>1</em>, 2, 4, 6, 8, and 24 h after injection of the endotoxin. Each patient's cardiovascular and overall clinical status was monitored over this period. The patients developed chills and rigors, myalgia, and fever between <em>1</em>-3 h. Tumor necrosis factor-alpha levels increased sharply at <em>1</em> h and peaked at 90 min, reaching the baseline concentration thereafter by 6 h. Interleukin-6 levels increased more gradually, peaking at 3 h and reaching the baseline concentration at 8 h. The procalcitonin concentration, which was undetectable (< <em>1</em>0 pg/<em>mL</em>) at 0, <em>1</em>, and 2 h, was detectable at 4 h and peaked at 6 h, maintaining a plateau through 8 and 24 h (4 ng/<em>mL</em>). There was no elevation of calcitonin concentrations, which remained below <em>1</em>0 pg/<em>mL</em>, the lowest sensitivity of the assay. Procalcitonin was measured by a two-antibody immunoradiometric assay specific for this peptide, with no cross-reactivity with calcitonin, katacalcin, or calcitonin gene-related peptide. We conclude that endotoxin induces the release of procalcitonin systemically, that this increase is not associated with an increase in calcitonin, and that the increase in procalcitonin associated with septicemia in patients may be mediated through the effect of endotoxin described here. Whether procalcitonin participates in the mechanisms underlying inflammation remains to be investigated.

We assessed the efficacy and safety of <em>1</em>0-d monotherapy with the orally administered CCR5 antagonist maraviroc in 63 HIV-<em>1</em>-positive individuals prescreened for the absence of CXCR4-using virus. Maximum reduction in viral load occurred at a median of <em>1</em>0-<em>1</em>5 d, with a mean reduction of>>or=<em>1</em>.6 log(<em>1</em>0) copies/<em>ml</em> at all twice daily doses>>or=<em>1</em>00 mg. These results provide proof of concept that CCR5 antagonism is a viable antiretroviral therapeutic approach.

OBJECTIVE

Controlling glycemia, blood pressure, and cholesterol is important for patients with diabetes. How best to achieve this goal is unknown.

OBJECTIVE

To compare Roux-en-Y gastric bypass with lifestyle and intensive medical management to achieve control of comorbid risk factors.

METHODS

A 12-month, 2-group unblinded randomized trial at 4 teaching hospitals in the United States and Taiwan involving 120 participants who had a hemoglobin A1c (HbA1c) level of 8.0% or higher, body mass index (BMI) between 30.0 and 39.9, C peptide level of more than 1.0 ng/mL, and type 2 diabetes for at least 6 months. The study began in April 2008.

METHODS

Lifestyle-intensive medical management intervention and Roux-en-Y gastric bypass surgery. Medications for hyperglycemia, hypertension, and dyslipidemia were prescribed according to protocol and surgical techniques that were standardized.

METHODS

Composite goal of HbA1c less than 7.0%, low-density lipoprotein cholesterol less than 100 mg/dL, and systolic blood pressure less than 130 mm Hg.

RESULTS

All 120 patients received the intensive lifestyle-medical management protocol and 60 were randomly assigned to undergo Roux-en-Y gastric bypass. After 12-months, 28 participants (49%; 95% CI, 36%-63%) in the gastric bypass group and 11 (19%; 95% CI, 10%-32%) in the lifestyle-medical management group achieved the primary end points (odds ratio [OR], 4.8; 95% CI, 1.9-11.7). Participants in the gastric bypass group required 3.0 fewer medications (mean, 1.7 vs 4.8; 95% CI for the difference, 2.3-3.6) and lost 26.1% vs 7.9% of their initial body weigh compared with the lifestyle-medical management group (difference, 17.5%; 95% CI, 14.2%-20.7%). Regression analyses indicated that achieving the composite end point was primarily attributable to weight loss. There were 22 serious adverse events in the gastric bypass group, including 1 cardiovascular event, and 15 in the lifestyle-medical management group. There were 4 perioperative complications and 6 late postoperative complications. The gastric bypass group experienced more nutritional deficiency than the lifestyle-medical management group.

CONCLUSIONS

In mild to moderately obese patients with type 2 diabetes, adding gastric bypass surgery to lifestyle and medical management was associated with a greater likelihood of achieving the composite goal. Potential benefits of adding gastric bypass surgery to the best lifestyle and medical management strategies of diabetes must be weighed against the risk of serious adverse events.

BACKGROUND

clinicaltrials.gov Identifier: NCT00641251.

In order to validate a maximal multistage 20-m shuttle run test for the prediction of VO2 max, 9<em>1</em> adults (32 females and 59 males, aged 27.3 +/- 9.2 and 24.8 +/- 5.5 year respectively and with mean VO2 max (+/- SD) of 39.3 +/- 8.3 and 5<em>1</em>.6 +/- 7.8 <em>ml</em> . kg-<em>1</em> . min-<em>1</em> respectively) performed the test and had VO2 max estimated by the retroextrapolation method (extrapolation to time zero of recovery of the exponential least squares regression of the first four 20-s recovery VO2 values). Starting at 8 km . h-<em>1</em> and increasing by 0.5 km . h-<em>1</em> every 2 min, the 20-m shuttle run test enabled prediction of the VO2 max (y, <em>ml</em> . kg-<em>1</em> . min-<em>1</em>) from the maximal speed (x, km . h-<em>1</em>) by means of the following regression equation: y = 5.857x - <em>1</em>9.458; r = 0.84 and SEE = 5.4. Later, the multistage protocol was slightly modified to its final version, in which the test started at stage 7 Met and continued with a <em>1</em> Met (3.5 <em>ml</em> O2 . kg-<em>1</em> . min-<em>1</em>) increment every 2 min. Twenty-five of the 9<em>1</em> subjects performed the 20-m shuttle test twice, once on a hard, low-friction surface (vinyl-asbestos tiles) and another time on a rubber floor, as well as a walking maximal multistage test on an inclined treadmill. There was no difference between the means of these tests or between the slopes of the VO2max - maximal speed regressions for the two types of surfaces. The 20-m shuttle run test and another maximal multistage field test involving continuous track running gave comparable results (r = 0.92, SEE = 2.6 <em>ml</em> O2 . kg-<em>1</em> . min-<em>1</em>, n = 70). Finally, test and retest of the 20-m shuttle run test also yielded comparable results (r = 0.975, SEE = 2.0 <em>ml</em> O2 . kg-<em>1</em> . min-<em>1</em>, n = 50). It is concluded that the 20-m shuttle run test is valid and reliable test for the prediction of the VO2 max of male and female adults, individually or in groups, on most gymnasium surfaces.

To present a summary of the 20<em>1</em>6 version of the European Association of Urology (EAU) - European Society for Radiotherapy & Oncology (ESTRO) - International Society of Geriatric Oncology (SIOG) Guidelines on the treatment of relapsing, metastatic, and castration-resistant prostate cancer (CRPC).

The working panel performed a literature review of the new data (20<em>1</em>3-20<em>1</em>5). The guidelines were updated, and the levels of evidence and/or grades of recommendation were added based on a systematic review of the literature.

Relapse after local therapy is defined by a rising prostate-specific antigen (PSA) level >0.2ng/<em>ml</em> following radical prostatectomy (RP) and >2ng/<em>ml</em> above the nadir after radiation therapy (RT). <em>1</em><em>1</em>C-choline positron emission tomography/computed tomography is of limited importance if PSA is (<em>1</em>.0ng/<em>ml</em>; bone scans and computed tomography can be omitted unless PSA is>><em>1</em>0ng/<em>ml</em>. Multiparametric magnetic resonance imaging and biopsy are important to assess biochemical failure following RT. Therapy for PSA relapse after RP includes salvage RT at PSA levels <0.5ng/<em>ml</em> and salvage RP, high-intensity focused ultrasound, cryosurgical ablation or salvage brachytherapy of the prostate in radiation failures. Androgen deprivation therapy (ADT) remains the basis for treatment of men with metastatic prostate cancer (PCa). However, docetaxel combined with ADT should be considered the standard of care for men with metastases at first presentation, provided they are fit enough to receive the drug. Follow-up of ADT should include analysis of PSA, testosterone levels, and screening for cardiovascular disease and metabolic syndrome. Level <em>1</em> evidence for the treatment of metastatic CRPC (mCRPC) includes, abiraterone acetate plus prednisone (AA/P), enzalutamide, radium 223 (Ra 223), docetaxel at 75 mg/m2 every 3 wk and sipuleucel-T. Cabazitaxel, AA/P, enzalutamide, and radium are approved for second-line treatment of CRPC following docetaxel. Zoledronic acid and denosumab can be used in men with mCRPC and osseous metastases to prevent skeletal-related complications.

The knowledge in the field of advanced and metastatic PCa and CRPC is changing rapidly. The 20<em>1</em>6 EAU-ESTRO-SIOG Guidelines on PCa summarise the most recent findings and advice for use in clinical practice. These PCa guidelines are the first endorsed by the European Society for Therapeutic Radiology and Oncology and the International Society of Geriatric Oncology and reflect the multidisciplinary nature of PCa management. A full version is available from the EAU office or online (http://uroweb.org/guideline/prostate-cancer/).

In men with a rise in their PSA levels after prior local treatment for prostate cancer only, it is important to balance overtreatment against further progression of the disease since survival and quality of life may never be affected in many of these patients. For patients diagnosed with metastatic castrate-resistant prostate cancer, several new drugs have become available which may provide a clear survival benefit but the optimal choice will have to be made on an individual basis.

OBJECTIVE

Glucagon-like peptide-<em>1</em> receptor (GLP-<em>1</em>R) agonists are used to treat type 2 diabetes, and transient GLP-<em>1</em> administration improved cardiac function in humans after acute myocardial infarction (MI) and percutaneous revascularization. However, the consequences of GLP-<em>1</em>R activation before ischemic myocardial injury remain unclear.

METHODS

We assessed the pathophysiology and outcome of coronary artery occlusion in normal and diabetic mice pretreated with the GLP-<em>1</em>R agonist liraglutide.

RESULTS

Male C57BL/6 mice were treated twice daily for 7 days with liraglutide or saline followed by induction of MI. Survival was significantly higher in liraglutide-treated mice. Liraglutide reduced cardiac rupture (<em>1</em>2 of 60 versus 46 of 60; P = 0.000<em>1</em>) and infarct size (2<em>1</em> +/- 2% versus 29 +/- 3%, P = 0.02) and improved cardiac output (<em>1</em>2.4 +/- 0.6 versus 9.7 +/- 0.6 ml/min; P = 0.002). Liraglutide also modulated the expression and activity of cardioprotective genes in the mouse heart, including Akt, GSK3beta, PPARbeta-delta, Nrf-2, and HO-<em>1</em>. The effects of liraglutide on survival were independent of weight loss. Moreover, liraglutide conferred cardioprotection and survival advantages over metformin, despite equivalent glycemic control, in diabetic mice with experimental MI. The cardioprotective effects of liraglutide remained detectable 4 days after cessation of therapy and may be partly direct, because liraglutide increased cyclic AMP formation and reduced the extent of caspase-3 activation in cardiomyocytes in a GLP-<em>1</em>R-dependent manner in vitro.

CONCLUSIONS

These findings demonstrate that GLP-<em>1</em>R activation engages prosurvival pathways in the normal and diabetic mouse heart, leading to improved outcomes and enhanced survival after MI in vivo.

OBJECTIVE

This study assessed longitudinal changes in body composition, fat distribution and energy balance in perimenopausal women. We hypothesized that total fat and abdominal body fat would increase at menopause due to decreased energy expenditure (EE) and declining estrogen, respectively.

METHODS

Observational, longitudinal study with annual measurements for 4 years.

METHODS

Healthy women (<em>1</em>03 Caucasian; 53 African-American), initially premenopausal. During follow-up, lack of menstruation for <em>1</em> year and follicle-stimulating hormone >30 mIU <em>ml</em>(-<em>1</em>) defined a subject as postmenopausal.

METHODS

Fat and lean mass (dual-energy X-ray absorptiometry), visceral (VAT) and subcutaneous abdominal fat (SAT) (computed tomography), dietary intake (4-day food record), serum sex hormones and physical activity (tri-axial accelerometry). Twenty-four hour EE was measured by whole-room calorimeter in a subset of 34 women at baseline and at year 4.

RESULTS

Body fat and weight increased significantly over time only in those women who became postmenopausal by year 4 (n=5<em>1</em>). All women gained SAT over time; however, only those who became postmenopausal had a significant increase in VAT. The postmenopausal group also exhibited a significant decrease in serum estradiol. Physical activity decreased significantly 2 years before menopause and remained low. Dietary energy, protein, carbohydrate and fiber intake were significantly higher 3-4 years before the onset of menopause compared with menopause onset. Twenty-four hour EE and sleeping EE decreased significantly with age; however, the decrease in sleeping EE was <em>1</em>.5-fold greater in women who became postmenopausal compared with premenopausal controls (-7.9 vs -5.3%). Fat oxidation decreased by 32% in women who became postmenopausal (P<0.05), but did not change in those who remained premenopausal.

CONCLUSIONS

Middle-aged women gained SAT with age, whereas menopause per se was associated with an increase in total body fat and VAT. Menopause onset is associated with decreased EE and fat oxidation that can predispose to obesity if lifestyle changes are not made.

BACKGROUND

The balance of risk and benefit from early neurosurgical intervention for conscious patients with superficial lobar intracerebral haemorrhage of 10-100 mL and no intraventricular haemorrhage admitted within 48 h of ictus is unclear. We therefore tested the hypothesis that early surgery compared with initial conservative treatment could improve outcome in these patients.

METHODS

In this international, parallel-group trial undertaken in 78 centres in 27 countries, we compared early surgical haematoma evacuation within 12 h of randomisation plus medical treatment with initial medical treatment alone (later evacuation was allowed if judged necessary). An automatic telephone and internet-based randomisation service was used to assign patients to surgery and initial conservative treatment in a 1:1 ratio. The trial was not masked. The primary outcome was a prognosis-based dichotomised (favourable or unfavourable) outcome of the 8 point Extended Glasgow Outcome Scale (GOSE) obtained by questionnaires posted to patients at 6 months. Analysis was by intention to treat. This trial is registered, number ISRCTN22153967.

RESULTS

307 of 601 patients were randomly assigned to early surgery and 294 to initial conservative treatment; 298 and 291 were followed up at 6 months, respectively; and 297 and 286 were included in the analysis, respectively. 174 (59%) of 297 patients in the early surgery group had an unfavourable outcome versus 178 (62%) of 286 patients in the initial conservative treatment group (absolute difference 3·7% [95% CI -4·3 to 11·6], odds ratio 0·86 [0·62 to 1·20]; p=0·367).

CONCLUSIONS

The STICH II results confirm that early surgery does not increase the rate of death or disability at 6 months and might have a small but clinically relevant survival advantage for patients with spontaneous superficial intracerebral haemorrhage without intraventricular haemorrhage.

BACKGROUND

UK Medical Research Council.

Men with high serum prostate specific antigen usually undergo transrectal ultrasound-guided prostate biopsy (TRUS-biopsy). TRUS-biopsy can cause side-effects including bleeding, pain, and infection. Multi-parametric magnetic resonance imaging (MP-MRI) used as a triage test might allow men to avoid unnecessary TRUS-biopsy and improve diagnostic accuracy.

We did this multicentre, paired-cohort, confirmatory study to test diagnostic accuracy of MP-MRI and TRUS-biopsy against a reference test (template prostate mapping biopsy [TPM-biopsy]). Men with prostate-specific antigen concentrations up to <em>1</em>5 ng/<em>mL</em>, with no previous biopsy, underwent <em>1</em>·5 Tesla MP-MRI followed by both TRUS-biopsy and TPM-biopsy. The conduct and reporting of each test was done blind to other test results. Clinically significant cancer was defined as Gleason score ≥4 + 3 or a maximum cancer core length 6 mm or longer. This study is registered on ClinicalTrials.gov, NCT0<em>1</em>29229<em>1</em>.

Between May <em>1</em>7, 20<em>1</em>2, and November 9, 20<em>1</em>5, we enrolled 740 men, 576 of whom underwent <em>1</em>·5 Tesla MP-MRI followed by both TRUS-biopsy and TPM-biopsy. On TPM-biopsy, 408 (7<em>1</em>%) of 576 men had cancer with 230 (40%) of 576 patients clinically significant. For clinically significant cancer, MP-MRI was more sensitive (93%, 95% CI 88-96%) than TRUS-biopsy (48%, 42-55%; p<0·000<em>1</em>) and less specific (4<em>1</em>%, 36-46% for MP-MRI vs 96%, 94-98% for TRUS-biopsy; p<0·000<em>1</em>). 44 (5·9%) of 740 patients reported serious adverse events, including 8 cases of sepsis.

Using MP-MRI to triage men might allow 27% of patients avoid a primary biopsy and diagnosis of 5% fewer clinically insignificant cancers. If subsequent TRUS-biopsies were directed by MP-MRI findings, up to <em>1</em>8% more cases of clinically significant cancer might be detected compared with the standard pathway of TRUS-biopsy for all. MP-MRI, used as a triage test before first prostate biopsy, could reduce unnecessary biopsies by a quarter. MP-MRI can also reduce over-diagnosis of clinically insignificant prostate cancer and improve detection of clinically significant cancer.

PROMIS is funded by the UK Government Department of Health, National Institute of Health Research-Health Technology Assessment Programme, (Project number 09/22/67). This project is also supported and partly funded by UCLH/UCL Biomedical Research Centre and The Royal Marsden and Institute for Cancer Research Biomedical Research Centre and is coordinated by the Medical Research Council Clinical Trials Unit (MRC CTU) at UCL. It is sponsored by University College London (UCL).

When Vicia hajastana Grossh. cells or protoplasts were cultured at a high population density (ca. 5000 cells or protoplasts/<em>ml</em>), they were able to grow in a mineral-salt solution supplemented with sucrose (or glucose), a few vitamins, and 2,4-dichlorophenoxyacetic acid. They were not able to survive when cultured at a low population density unless the medium was supplemented with zeatin, naphthalene-<em>1</em>-acetic acid, nucleic-acid bases, amino acids, other sugars, sugar alcohols, and organic acids. Vicia cells were able to grow at an initial population density of 25-50 cells/<em>ml</em> in this defined medium. The population density could be lowered to <em>1</em>-2 cells/<em>ml</em> with good growth when the mineral-salt medium was enriched with organic acids, sugars, sugar alcohols, coconut water, and casamino acids. The protoplasts also grew best in a medium enriched with these supplements. Three individual protoplasts were isolated and each one was cultured in a separate dish containing 4 <em>ml</em> of this medium. Within 30-40 days, each one had grown indefinitely and formed a mass of cells (ca. <em>1</em>0(7)).