A panel of human monoclonal antibody Fab fragments has been generated against the surface glycoprotein gp<em>1</em>20 of type <em>1</em> human immunodeficiency virus (HIV) by antigen selection from a random combinatorial library expressed on the surface of filamentous phage. The library was prepared from 5 <em>ml</em> of bone marrow from an asymptomatic individual who has been HIV-positive for 6 years. The antibodies have high affinity for antigen (mostly with affinity constants of greater than <em>1</em>0(8) M-<em>1</em>) and notable sequence diversity. Given appropriate donor selection, the methods described should allow the generation of antibodies for the evaluation of passive immunization as a therapy for AIDS.

BACKGROUND

The safety and short-term efficacy of laparoscopic surgery for rectal cancer after preoperative chemoradiotherapy has not been demonstrated. The aim of the randomised Comparison of Open versus laparoscopic surgery for mid and low REctal cancer After Neoadjuvant chemoradiotherapy (COREAN) trial was to compare open surgery with laparoscopic surgery for mid or low rectal cancer after neoadjuvant chemoradiotherapy.

METHODS

Between April 4, 2006, and Aug 26, 2009, patients with cT3N0-2 mid or low rectal cancer without distant metastasis after preoperative chemoradiotherapy were enrolled at three tertiary-referral hospitals. Patients were randomised <em>1</em>:<em>1</em> to receive either open surgery (n=<em>1</em>70) or laparoscopic surgery (n=<em>1</em>70), stratified according to sex and preoperative chemotherapy regimen. Short-term outcomes assessed were involvement of the circumferential resection margin, macroscopic quality of the total mesorectal excision specimen, number of harvested lymph nodes, recovery of bowel function, perioperative morbidity, postoperative pain, and quality of life. Analyses were based on the intention-to-treat population. Patients continue to be followed up for the primary outcome (3-year disease-free survival). This study is registered with ClinicalTrials.gov, number NCT0047095<em>1</em>.

RESULTS

Two patients (<em>1</em>.2%) in the laparoscopic group were converted to open surgery, but were included in the laparoscopic group for analyses. Estimated blood loss was less in the laparoscopic group than in the open group (median 2<em>1</em>7.5 mL [<em>1</em>50.0-400.0] in the open group vs 200.0 mL [<em>1</em>00.0-300.0] in the laparoscopic group, p=0.006), although surgery time was longer in the laparoscopic group (mean 244.9 min [SD 75.4] vs <em>1</em>97.0 min [62.9], p<0.000<em>1</em>). Involvement of the circumferential resection margin, macroscopic quality of the total mesorectal excision specimen, number of harvested lymph nodes, and perioperative morbidity did not differ between the two groups. The laparoscopic surgery group showed earlier recovery of bowel function than the open surgery group (time to pass first flatus, median 38.5 h [23.0-53.0] vs 60.0 h [43.0-73.0], p<0.000<em>1</em>; time to resume a normal diet, 85.0 h [66.0-95.0] vs 93.0 h [86.0-<em>1</em>2<em>1</em>.0], p<0.000<em>1</em>; time to first defecation, 96.5 h [70.0-<em>1</em>25.0] vs <em>1</em>23 h [94.0-<em>1</em>56.0], p<0.000<em>1</em>). The total amount of morphine used was less in the laparoscopic group than in the open group (median <em>1</em>07.2 mg [80.0-<em>1</em>50.0] vs <em>1</em>56.9 mg [<em>1</em><em>1</em>7.0-<em>1</em>85.2], p<0.000<em>1</em>). 3 months after proctectomy or ileostomy takedown, the laparoscopic group showed better physical functioning score than the open group (0.50<em>1</em> [n=<em>1</em>22] vs -4.970 [n=<em>1</em>28], p=0.0073), less fatigue (-5.659 [n=<em>1</em>22] vs 0.098 [n=<em>1</em>29], p=0.0206), and fewer micturition (-2.583 [n=<em>1</em>22] vs 4.725 [n=<em>1</em>29], p=0.0002), gastrointestinal (-0.400 [n=<em>1</em>22] vs 4.33<em>1</em> [n=<em>1</em>29], p=0.0<em>1</em>02), and defecation problems (0.535 [n=<em>1</em>03] vs 5.327 [n=99], p=0.0<em>1</em>84) in repeated measures analysis of covariance, adjusted for baseline values.

CONCLUSIONS

Laparoscopic surgery after preoperative chemoradiotherapy for mid or low rectal cancer is safe and has short-term benefits compared with open surgery; the quality of oncological resection was equivalent.

We examined the effect of brefeldin A, an antiviral antibiotic, on protein synthesis, intracellular processing, and secretion in primary culture of rat hepatocytes. The secretion was strongly blocked by the drug at <em>1</em> microgram/<em>ml</em> and higher concentrations, while the protein synthesis was maintained fairly well. Pulse-chase experiments with [35S]methionine demonstrated that brefeldin A completely blocked the proteolytic conversion of proalbumin to serum albumin up to 60 min of chase, although its conversion was observed as early as 20 min in the control cells. The drug also inhibited the terminal glycosylation of oligosaccharide chains of alpha <em>1</em>-protease inhibitor and haptoglobin. These two modifications have been shown to occur at the trans region of the Golgi complex. The drug, however, had no effect on the proteolytic processing of the haptoglobin proform which takes place within the endoplasmic reticulum. Such an effect by brefeldin A is very similar with that induced by the carboxylic ionophore monensin. However, in contrast to evidence that monensin causes a delayed secretion of the unprocessed forms of these proteins, brefeldin A allowed the completely processed forms to be secreted after a prolonged accumulation of the unprocessed forms. Morphological observations demonstrated that the endoplasmic reticulum was markedly dilated by treatment with the drug at <em>1</em>0 micrograms/<em>ml</em> which continuously blocked the secretion. On the other hand, brefeldin A caused no inhibitory effect on the endocytic pathway as judged by cellular uptake and degradation of <em>1</em>25I-asialofetuin. These results indicate that brefeldin A is a unique agent which primarily impedes protein transport from the endoplasmic reticulum to the Golgi complex by a mechanism different from those considered for other secretion-blocking agents so far reported.

We have evaluated the role of nitric oxide (NO) on the activity of the constitutive and induced forms of cyclooxygenase (COX; COX-<em>1</em> and COX-2, respectively). Induction of NO synthase (NOS) and COX (COX-2) in the mouse macrophage cell line RAW264.7 by Escherichia coli lipopolysaccharide (<em>1</em> microgram/<em>ml</em>, <em>1</em>8 h) caused an increase in the release of nitrite (NO2-) and prostaglandin E2 (PGE2), products of NOS and COX, respectively. Production of both NO2- and PGE2 was blocked by the NOS inhibitors NG-monomethyl-L-arginine or aminoguanidine. The effects of NG-monomethyl-L-arginine or aminoguanidine were reversed by coincubation with L-Arg, the precursor for NO synthesis, but not by D-Arg. RAW264.7 cells stimulated for <em>1</em>8 h with lipopolysaccharide in L-Arg-free medium (to reduce NO generation by the endogenous NOS pathway) failed to release NO2- and accumulated at least 4-fold less PGE2 when compared to cells in the presence of L-Arg. PGE2 production elicited by a <em>1</em>5-min arachidonic acid treatment of lipopolysaccharide-induced RAW264.7 cells in L-Arg-deficient medium was decreased 3-fold when compared to the release obtained with cells induced in medium containing L-Arg. To examine the NO activation of the induced form of COX in the absence of an endogenous L-Arg, human fetal fibroblasts were first stimulated for <em>1</em>8 h with interleukin <em>1</em> beta. These cells released PGE2 but not NO2-, consistent with the induction of COX but not NOS in the fibroblast. Exogenous NO either as a gaseous solution or released by a NO donor, sodium nitroprusside or glyceryl trinitrate, increased COX activity in the interleukin <em>1</em> beta-stimulated fibroblasts by 5-fold; these effects were abolished by coincubation with hemoglobin (<em>1</em>0 microM), which binds and inactivates NO, but not by methylene blue, an inhibitor of the soluble guanylate cyclase. Furthermore, sodium nitroprusside (0.25-<em>1</em> mM) increased arachidonic acid-stimulated PGE2 production by murine recombinant COX-<em>1</em> and COX-2. These results demonstrate that NO enhances COX activity through a mechanism independent of cGMP and suggest that, in conditions in which both the NOS and COX systems are present, there is an NO-mediated increase in the production of proinflammatory prostaglandins that may result in an exacerbated inflammatory response. The data suggest that NO directly interacts with COX to cause an increase in the enzymatic activity.

The mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expression is unknown. To determine whether changes in isoprenoid synthesis affects eNOS expression, human endothelial cells were treated with the HMG-CoA reductase inhibitor, mevastatin (<em>1</em>-<em>1</em>0 microM), in the presence of L-mevalonate (200 microM), geranylgeranylpyrophosphate (GGPP, <em>1</em>-<em>1</em>0 microM), farnesylpyrophosphate (FPP, 5-<em>1</em>0 microM), or low density lipoprotein (LDL, <em>1</em> mg/<em>ml</em>). Mevastatin increased eNOS mRNA and protein levels by 305 +/- <em>1</em>5% and <em>1</em>80 +/- <em>1</em><em>1</em>%, respectively. Co-treatment with L-mevalonate or GGPP, but not FPP or LDL, reversed mevastatin's effects. Because Rho GTPases undergo geranylgeranyl modification, we investigated whether Rho regulates eNOS expression. Immunoblot analyses and [35S]GTPgammaS-binding assays revealed that mevastatin inhibited Rho membrane translocation and GTP binding activity by 60 +/- 5% and 78 +/- 6%, both of which were reversed by co-treatment with GGPP but not FPP. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase (50 microg/<em>ml</em>) or by overexpression of a dominant-negative N<em>1</em>9RhoA mutant increased eNOS expression. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-<em>1</em> (200 ng/<em>ml</em>) decreased eNOS expression. These findings indicate that Rho negatively regulates eNOS expression and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is necessary for its membrane-associated activity.

BACKGROUND

The Women and Infants Transmission Study is a prospective natural history study that has been enrolling HIV-<em>1</em>-infected pregnant women and their infants since <em>1</em>989.

OBJECTIVE

To evaluate the impact of different antiretroviral regimens on perinatal HIV-<em>1</em> transmission at the population level.

METHODS

Prospective cohort study. Plasma HIV-<em>1</em> RNA levels were serially measured in <em>1</em>542 HIV-<em>1</em>-infected women with singleton live births between January <em>1</em>990 and June 2000.

METHODS

HIV-<em>1</em> status of the infant.

RESULTS

HIV-<em>1</em> transmission was 20.0% (95% confidence interval [CI], <em>1</em>6.<em>1</em>%-23.9%) for 396 women who not receiving prenatal antiretroviral therapy; <em>1</em>0.4% (95% CI, 8.2%-<em>1</em>2.6%) for 7<em>1</em>0 receiving zidovudine monotherapy; 3.8% (95% CI, <em>1</em>.<em>1</em>%-6.5%) for <em>1</em>86 receiving dual antiretroviral therapy with no or one highly active drug (Multi-ART); and <em>1</em>.2% (95% CI, 0-2.5%) for 250 receiving highly active antiretroviral therapy (HAART). Transmission also varied by maternal delivery HIV RNA level: <em>1</em>.0% for <400; 5.3% for 400 to 3499; 9.3% for 3500 to 9999; <em>1</em>4.7% for <em>1</em>0,000 to 29,999; and 23.4% for >30,000 copies/mL (p =.000<em>1</em> for trend). The odds of transmission increased 2.4-fold (95% CI, <em>1</em>.7-3.5) for every log<em>1</em>0 increase in delivery viral load. In multivariate analyses adjusting for maternal viral load, duration of therapy, and other factors, the odds ratio for transmission for women receiving Multi-ART and HAART compared with those receiving ZDV monotherapy was 0.30 (95% CI, 0.09-<em>1</em>.02) and 0.27 (95% CI, 0.08-0.94), respectively.

CONCLUSIONS

Levels of HIV-<em>1</em> RNA at delivery and prenatal antiretroviral therapy were independently associated with transmission. The protective effect of therapy increased with the complexity and duration of the regimen. HAART was associated with the lowest rates of transmission.

Adiponectin has antilipogenic and anti-inflammatory effects, while tumor necrosis factor alpha (TNF-alpha) reduces insulin sensitivity and has proinflammatory effects. We examined (<em>1</em>) the extent to which hypoadiponectinemia and TNF-alpha activation are features of nonalcoholic steatohepatitis (NASH) and (2) whether serum levels of these markers correlate with the severity of histological changes in <em>1</em>09 subjects with nonalcoholic fatty liver disease (NAFLD), including 80 with NASH and 29 with simple steatosis. By multivariate analysis, subjects with NASH had reduced adiponectin level and increased TNF-alpha and soluble TNF receptor 2 (sTNFR2)-but not leptin levels, compared with controls matched by age, sex, and body mass index; these differences were independent of the increased insulin resistance (by homeostasis model [HOMA-IR]) in NASH. When compared with simple steatosis, NASH was associated with lower adiponectin levels and higher HOMA-IR, but there were no significant differences in the levels of TNF-alpha and sTNFR2. The majority of subjects with steatohepatitis (77%) had adiponectin levels less than <em>1</em>0 microg/<em>mL</em> and HOMA-IR greater than 3 units, but only 33% of those with pure steatosis had these findings. HOMA-IR and low serum adiponectin were also independently associated with increased grades of hepatic necroinflammation. In conclusion, hypoadiponectinemia is a feature of NASH independent of insulin resistance. Reduced adiponectin level is associated with more extensive necroinflammation and may contribute to the development of necroinflammatory forms of NAFLD.

BACKGROUND

Patients with human immunodeficiency virus (HIV) infection need lifelong medical care, but many do not remain in care. The effect of poor retention in care on survival is not known, and we sought to quantify that relationship.

METHODS

We conducted a retrospective cohort study involving persons newly identified as having HIV infection during <em>1</em>997-<em>1</em>998 at any United States Department of Veterans Affairs hospital or clinic who started antiretroviral therapy after <em>1</em> January <em>1</em>997. To be included in the study, patients had to have seen a clinician at least once after receiving their first antiretroviral prescription and to have survived for at least <em>1</em> year. Patients were divided into 4 groups on the basis of the number of quarters in that year during which they had at least <em>1</em> HIV primary care visit. Survival was measured through 2002. Because data were available for only a small number of women, female patients were excluded from the study.

RESULTS

A total of 26<em>1</em>9 men were followed up for a mean of >4 years each. The median baseline CD4(+) cell count and median log(<em>1</em>0) plasma HIV concentration were 228x<em>1</em>0(6) cells/L and 4.58 copies/mL, respectively. Thirty-six percent of the patients had visits in <4 quarters, and <em>1</em>6% died during follow-up. In Cox multivariate regression analysis, compared with persons with visits in all 4 quarters during the first year, the adjusted hazard ratio of death was <em>1</em>.42 (95% confidence interval, <em>1</em>.<em>1</em><em>1</em>-<em>1</em>.83; P<.0<em>1</em>), <em>1</em>.67 (95% confidence interval, <em>1</em>.24-2.25; P<.00<em>1</em>), and <em>1</em>.95 (95% confidence interval, <em>1</em>.37-2.78; P<.00<em>1</em>) for persons with visits in 3 quarters, 2 quarters, and <em>1</em> quarter, respectively.

CONCLUSIONS

Even in a system with few financial barriers to care, a substantial portion of HIV-infected patients have poor retention in care. Poor retention in care predicts poorer survival with HIV infection. Retaining persons in care may improve survival, and optimal methods to retain patients need to be defined.

Ghrelin, a circulating growth hormone-releasing peptide derived from the stomach, stimulates food intake. The lowest systemically effective orexigenic dose of ghrelin was investigated and the resulting plasma ghrelin concentration was compared with that during fasting. The lowest dose of ghrelin that produced a significant stimulation of feeding after intraperitoneal injection was <em>1</em> nmol. The plasma ghrelin concentration after intraperitoneal injection of <em>1</em> nmol of ghrelin (2.83 +/- 0.<em>1</em>3 pmol/<em>ml</em> at 60 min postinjection) was not significantly different from that occurring after a 24-h fast (2.79 +/- 0.32 pmol/<em>ml</em>). After microinjection into defined hypothalamic sites, ghrelin (30 pmol) stimulated food intake most markedly in the arcuate nucleus (Arc) (0-<em>1</em> h food intake, 427 +/- 43% of control; P < 0.00<em>1</em> vs. control, P < 0.0<em>1</em> vs. all other nuclei), which is potentially accessible to the circulation. After chronic systemic or intracerebroventricular (ICV) administration of ghrelin for 7 days, cumulative food intake was increased (intraperitoneal ghrelin <em>1</em>3.6 +/- 3.4 g greater than saline-treated, P < 0.0<em>1</em>; ICV ghrelin <em>1</em>9.6 +/- 5.5 g greater than saline-treated, P < 0.05). This was associated with excess weight gain (intraperitoneal ghrelin 2<em>1</em>.7 +/- <em>1</em>.4 g vs. saline <em>1</em>0.6 +/- <em>1</em>.9 g, P < 0.00<em>1</em>; ICV ghrelin <em>1</em>5.3 +/- 4.3 g vs. saline 2.2 +/- 3.8 g, P < 0.05) and adiposity. These data provide evidence that ghrelin is important in long-term control of food intake and body weight and that circulating ghrelin at fasting concentrations may stimulate food intake.

BACKGROUND

Long-term hormone therapy has been the standard of care for advanced prostate cancer since the <em>1</em>940s. STAMPEDE is a randomised controlled trial using a multiarm, multistage platform design. It recruits men with high-risk, locally advanced, metastatic or recurrent prostate cancer who are starting first-line long-term hormone therapy. We report primary survival results for three research comparisons testing the addition of zoledronic acid, docetaxel, or their combination to standard of care versus standard of care alone.

METHODS

Standard of care was hormone therapy for at least 2 years; radiotherapy was encouraged for men with N0M0 disease to November, 20<em>1</em><em>1</em>, then mandated; radiotherapy was optional for men with node-positive non-metastatic (N+M0) disease. Stratified randomisation (via minimisation) allocated men 2:<em>1</em>:<em>1</em>:<em>1</em> to standard of care only (SOC-only; control), standard of care plus zoledronic acid (SOC + ZA), standard of care plus docetaxel (SOC + Doc), or standard of care with both zoledronic acid and docetaxel (SOC + ZA + Doc). Zoledronic acid (4 mg) was given for six 3-weekly cycles, then 4-weekly until 2 years, and docetaxel (75 mg/m(2)) for six 3-weekly cycles with prednisolone <em>1</em>0 mg daily. There was no blinding to treatment allocation. The primary outcome measure was overall survival. Pairwise comparisons of research versus control had 90% power at 2·5% one-sided α for hazard ratio (HR) 0·75, requiring roughly 400 control arm deaths. Statistical analyses were undertaken with standard log-rank-type methods for time-to-event data, with hazard ratios (HRs) and 95% CIs derived from adjusted Cox models. This trial is registered at ClinicalTrials.gov (NCT00268476) and ControlledTrials.com (ISRCTN788<em>1</em>8544).

RESULTS

2962 men were randomly assigned to four groups between Oct 5, 2005, and March 3<em>1</em>, 20<em>1</em>3. Median age was 65 years (IQR 60-7<em>1</em>). <em>1</em>8<em>1</em>7 (6<em>1</em>%) men had M+ disease, 448 (<em>1</em>5%) had N+/X M0, and 697 (24%) had N0M0. <em>1</em>65 (6%) men were previously treated with local therapy, and median prostate-specific antigen was 65 ng/mL (IQR 23-<em>1</em>84). Median follow-up was 43 months (IQR 30-60). There were 4<em>1</em>5 deaths in the control group (347 [84%] prostate cancer). Median overall survival was 7<em>1</em> months (IQR 32 to not reached) for SOC-only, not reached (32 to not reached) for SOC + ZA (HR 0·94, 95% CI 0·79-<em>1</em>·<em>1</em><em>1</em>; p=0·450), 8<em>1</em> months (4<em>1</em> to not reached) for SOC + Doc (0·78, 0·66-0·93; p=0·006), and 76 months (39 to not reached) for SOC + ZA + Doc (0·82, 0·69-0·97; p=0·022). There was no evidence of heterogeneity in treatment effect (for any of the treatments) across prespecified subsets. Grade 3-5 adverse events were reported for 399 (32%) patients receiving SOC, <em>1</em>97 (32%) receiving SOC + ZA, 288 (52%) receiving SOC + Doc, and 269 (52%) receiving SOC + ZA + Doc.

CONCLUSIONS

Zoledronic acid showed no evidence of survival improvement and should not be part of standard of care for this population. Docetaxel chemotherapy, given at the time of long-term hormone therapy initiation, showed evidence of improved survival accompanied by an increase in adverse events. Docetaxel treatment should become part of standard of care for adequately fit men commencing long-term hormone therapy.

BACKGROUND

Cancer Research UK, Medical Research Council, Novartis, Sanofi-Aventis, Pfizer, Janssen, Astellas, NIHR Clinical Research Network, Swiss Group for Clinical Cancer Research.

A simple, rapid and inexpensive method for the measurement of hydrogen peroxide (H2O2) produced by cells in culture is described. The assay is based on the horseradish peroxidase (HRPO)-mediated oxidation of phenol red by H2O2 which results in the formation of a compound demonstrating increased absorbance at 6<em>1</em>0 nm. A linear relationship between absorbance at 6<em>1</em>0 nm and concentration of H2O2 was found in the <em>1</em>--60 microM (<em>1</em>--60 nmoles/<em>ml</em>) range. Due to the non-toxic character of phenol red and HRPO, the assay permits measurement of H2O2 production and release by macrophages for time intervals of 5--60 min under regular tissue culture conditions. Using this assay, the ability of a number of agents to induce H2O2 release by guinea pig peritoneal macrophages was demonstrated. These agents were: phorbol myristate acetate (PMA), opsonized zymosan, concanavalin A (Con A), wheat germ agglutinin (WGA), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and A23<em>1</em>87.

Development of an in vitro living cell-based model of the intestine that mimics the mechanical, structural, absorptive, transport and pathophysiological properties of the human gut along with its crucial microbial symbionts could accelerate pharmaceutical development, and potentially replace animal testing. Here, we describe a biomimetic 'human gut-on-a-chip' microdevice composed of two microfluidic channels separated by a porous flexible membrane coated with extracellular matrix (ECM) and lined by human intestinal epithelial (Caco-2) cells that mimics the complex structure and physiology of living intestine. The gut microenvironment is recreated by flowing fluid at a low rate (30 <em>μL</em> h(-<em>1</em>)) producing low shear stress (0.02 dyne cm(-2)) over the microchannels, and by exerting cyclic strain (<em>1</em>0%; 0.<em>1</em>5 Hz) that mimics physiological peristaltic motions. Under these conditions, a columnar epithelium develops that polarizes rapidly, spontaneously grows into folds that recapitulate the structure of intestinal villi, and forms a high integrity barrier to small molecules that better mimics whole intestine than cells in cultured in static Transwell models. In addition, a normal intestinal microbe (Lactobacillus rhamnosus GG) can be successfully co-cultured for extended periods >><em>1</em> week) on the luminal surface of the cultured epithelium without compromising epithelial cell viability, and this actually improves barrier function as previously observed in humans. Thus, this gut-on-a-chip recapitulates multiple dynamic physical and functional features of human intestine that are critical for its function within a controlled microfluidic environment that is amenable for transport, absorption, and toxicity studies, and hence it should have great value for drug testing as well as development of novel intestinal disease models.

Data are presented demonstrating that DNA damage leads to specific post-translational modifications of p53 protein. Using two-dimensional peptide mapping of in vivo radiolabeled p53 tryptic phosphopeptides, recombinant truncated p53 protein, and synthetic p53 tryptic peptides, a unique p53 phosphopeptide was identified after exposure of <em>ML</em>-<em>1</em> cells to ionizing irradiation. This peptide represents the first 24 amino acids of p53 and contains three phosphorylated serine residues. A specific p53 phosphopeptide antibody identified serine-<em>1</em>5 as one of the two serines in p53 that becomes phosphorylated following DNA damage induced by either ionizing irradiation (IR) or ultraviolet (UV) irradiation in multiple cell types. IR-induced phosphorylation of p53 does not affect the kinetics of p53 binding to or dissociating from DNA as assessed by electrophoretic mobility-shift assays. However, p53 phosphorylation induced by DNA damage correlates with enhanced transcription of downstream p53 target genes. Low levels of phosphoserine-<em>1</em>5 p53 are detectable within 6 hr after IR in AT cells, whereas lymphoblasts from normal individuals exhibit this modification within <em>1</em> hr. In contrast, phosphorylation of p53 on serine-<em>1</em>5 is similar in normal and AT cells after UV irradiation. Our results indicate that p53 is phosphorylated in response to DNA damage, that this de novo phosphorylation may be involved in the subsequent induction and activation of p53, and that although ATM affects the kinetics of p53 phosphorylation after IR, it is not absolutely required for phosphorylation of p53 on serine-<em>1</em>5.

BACKGROUND

Wasting in chronic heart failure (CHF) has long been known but is little investigated. We sought to find out whether the cachectic state in CHF provides additional prognostic information about all-cause mortality.

METHODS

Between June, <em>1</em>993, and May, <em>1</em>995, we studied <em>1</em>7<em>1</em> consecutive patients with CHF (mean age 60 years [SD <em>1</em><em>1</em>; range 27-86]; <em>1</em>7 female). We assessed exercise capacity (peak oxygen consumption; mean <em>1</em>7.5 <em>mL</em> kg-<em>1</em> min-<em>1</em> [6.7]), functional status (New York Heart Association [NYHA] class: 2<em>1</em> class I, 63 class II, 68 class III, <em>1</em>9 class IV), and left-ventricular ejection fraction (mean 30% [SD <em>1</em>5]; n = <em>1</em><em>1</em>5). The cachectic status was defined prospectively as a non-intentional documented weight loss of at least 7.5% of previous normal weight (28 patients; range 9-36% or 6-30 kg) during at least 6 months. The Cox proportional-hazards model was used to assess the association of variables with survival, and Kaplan-Meier cumulative survival plots were constructed to estimate the influence of risk factors.

RESULTS

At the end of follow-up in November, <em>1</em>996, 49 patients had died (after a mean 324 days [SD 283]). The mean follow-up of the survivors was 834 days (SD <em>1</em>86; range 549-<em>1</em>269). The cachectic state was predictive of <em>1</em>8-month mortality independent of age, NYHA class, left-ventricular ejection fraction, and peak oxygen consumption. Mortality in the cachectic patients (n = 28) was <em>1</em>8% at 3 months, 29% at 6 months, 39% at <em>1</em>2 months, and 50% at <em>1</em>8 months. Patients who had a peak oxygen consumption below <em>1</em>4 <em>mL</em> kg-<em>1</em> min-<em>1</em> (n = 53) had mortality at 3, 6, <em>1</em>2, and <em>1</em>8 months of <em>1</em>9%, 30%, 40%, and 5<em>1</em>%. <em>1</em>8-month survival was 23% (95% CI 0-46) for the <em>1</em>3 patients with both of these risk factors (cachexia and low peak oxygen consumption) compared with 93% (88-98) in those (n = <em>1</em>03) with neither risk factor (p < 0.000<em>1</em>).

CONCLUSIONS

The cachectic state is a strong independent risk factor for mortality in patients with CHF. Combined with a low peak oxygen consumption, it identifies a subset of patients at extremely high risk of death. Assessment of cachexia should be included in transplant programmes and studies that investigate the effect of interventions by survival analyses.

Erythropoietin (EPO) promotes neuronal survival after hypoxia and other metabolic insults by largely unknown mechanisms. Apoptosis and necrosis have been proposed as mechanisms of cellular demise, and either could be the target of actions of EPO. This study evaluates whether antiapoptotic mechanisms can account for the neuroprotective actions of EPO. Systemic administration of EPO (5,000 units/kg of body weight, i.p.) after middle-cerebral artery occlusion in rats dramatically reduces the volume of infarction 24 h later, in concert with an almost complete reduction in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling of neurons within the ischemic penumbra. In both pure and mixed neuronal cultures, EPO (0.<em>1</em>--<em>1</em>0 units/<em>ml</em>) also inhibits apoptosis induced by serum deprivation or kainic acid exposure. Protection requires pretreatment, consistent with the induction of a gene expression program, and is sustained for 3 days without the continued presence of EPO. EPO (0.3 units/<em>ml</em>) also protects hippocampal neurons against hypoxia-induced neuronal death through activation of extracellular signal-regulated kinases and protein kinase Akt-<em>1</em>/protein kinase B. The action of EPO is not limited to directly promoting cell survival, as EPO is trophic but not mitogenic in cultured neuronal cells. These data suggest that inhibition of neuronal apoptosis underlies short latency protective effects of EPO after cerebral ischemia and other brain injuries. The neurotrophic actions suggest there may be longer-latency effects as well. Evaluation of EPO, a compound established as clinically safe, as neuroprotective therapy in acute brain injury is further supported.

The roles of slow antibiotic penetration, oxygen limitation, and low metabolic activity in the tolerance of Pseudomonas aeruginosa in biofilms to killing by antibiotics were investigated in vitro. Tobramycin and ciprofloxacin penetrated biofilms but failed to effectively kill the bacteria. Bacteria in colony biofilms survived prolonged exposure to either <em>1</em>0 micro g of tobramycin <em>ml</em>(-<em>1</em>)or <em>1</em>.0 micro g of ciprofloxacin <em>ml</em>(-<em>1</em>). After <em>1</em>00 h of antibiotic treatment, during which the colony biofilms were transferred to fresh antibiotic-containing plates every 24 h, the log reduction in viable cell numbers was only 0.49 +/- 0.<em>1</em>8 for tobramycin and <em>1</em>.42 +/- 0.03 for ciprofloxacin. Antibiotic permeation through colony biofilms, indicated by a diffusion cell bioassay, demonstrated that there was no acceleration in bacterial killing once the antibiotics penetrated the biofilms. These results suggested that limited antibiotic diffusion is not the primary protective mechanism for these biofilms. Transmission electron microscopic observations of antibiotic-affected cells showed lysed, vacuolated, and elongated cells exclusively near the air interface in antibiotic-treated biofilms, suggesting a role for oxygen limitation in protecting biofilm bacteria from antibiotics. To test this hypothesis, a microelectrode analysis was performed. The results demonstrated that oxygen penetrated 50 to 90 micro m into the biofilm from the air interface. This oxic zone correlated to the region of the biofilm where an inducible green fluorescent protein was expressed, indicating that this was the active zone of bacterial metabolic activity. These results show that oxygen limitation and low metabolic activity in the interior of the biofilm, not poor antibiotic penetration, are correlated with antibiotic tolerance of this P. aeruginosa biofilm system.

BACKGROUND

Erythropoietin (EPO) and its receptor play a major role in embryonic brain, are weakly expressed in normal postnatal/adult brain and up-regulated upon metabolic stress. EPO protects neurons from hypoxic/ ischemic injury. The objective of this trial is to study the safety and efficacy of recombinant human EPO (rhEPO) for treatment of ischemic stroke in man.

METHODS

The trial consisted of a safety part and an efficacy part. In the safety study, 13 patients received rhEPO intravenously (3.3 X 10(4) IU/50 ml/30 min) once daily for the first 3 days after stroke. In the double-blind randomized proof-of-concept trial, 40 patients received either rhEPO or saline. Inclusion criteria were age <80 years, ischemic stroke within the middle cerebral artery territory confirmed by diffusion-weighted MRI, symptom onset <8 hr before drug administration, and deficits on stroke scales. The study endpoints were functional outcome at day 30 (Barthel Index, modified Rankin scale), NIH and Scandinavian stroke scales, evolution of infarct size (sequential MRI evaluation using diffusion-weighted [DWI] and fluid-attenuated inversion recovery sequences [FLAIR]) and the damage marker S100ss.

RESULTS

No safety concerns were identified. Cerebrospinal fluid EPO increased to 60-100 times that of nontreated patients, proving that intravenously administered rhEPO reaches the brain. In the efficacy trial, patients received rhEPO within 5 hr of onset of symptoms (median, range 2:40-7:55). Admission neurologic scores and serum S100beta concentrations were strong predictors ofoutcome. Analysis of covariance controlled for these two variables indicated that rhEPO treatment was associated with an improvement in follow-up and outcome scales. A strong trend for reduction in infarct size in rhEPO patients as compared to controls was observed by MRI.

CONCLUSIONS

Intravenous high-dose rhEPO is well tolerated in acute ischemic stroke and associated with an improvement in clinical outcome at 1 month. A larger scale clinical trial is warranted.

Fully exploiting the properties of graphene will require a method for the mass production of this remarkable material. Two main routes are possible: large-scale growth or large-scale exfoliation. Here, we demonstrate graphene dispersions with concentrations up to approximately 0.0<em>1</em> mg <em>ml</em>(-<em>1</em>), produced by dispersion and exfoliation of graphite in organic solvents such as N-methyl-pyrrolidone. This is possible because the energy required to exfoliate graphene is balanced by the solvent-graphene interaction for solvents whose surface energies match that of graphene. We confirm the presence of individual graphene sheets by Raman spectroscopy, transmission electron microscopy and electron diffraction. Our method results in a monolayer yield of approximately <em>1</em> wt%, which could potentially be improved to 7-<em>1</em>2 wt% with further processing. The absence of defects or oxides is confirmed by X-ray photoelectron, infrared and Raman spectroscopies. We are able to produce semi-transparent conducting films and conducting composites. Solution processing of graphene opens up a range of potential large-area applications, from device and sensor fabrication to liquid-phase chemistry.

Patients with chronic hepatitis B virus (HBV) infection who develop antiviral resistance lose benefits of therapy and may be predisposed to further resistance. Entecavir (ETV) resistance (ETVr) results from HBV reverse transcriptase substitutions at positions T<em>1</em>84, S202, or M250, which emerge in the presence of lamivudine (LVD) resistance substitutions M204I/V +/- L<em>1</em>80M. Here, we summarize results from comprehensive resistance monitoring of patients with HBV who were continuously treated with ETV for up to 5 years. Monitoring included genotypic analysis of isolates from all patients at baseline and when HBV DNA was detectable by polymerase chain reaction >> or = 300 copies/<em>mL</em>) from Years <em>1</em> through 5. In addition, genotyping was performed on isolates from patients experiencing virologic breakthrough >> or = <em>1</em> log(<em>1</em>0) rise in HBV DNA). In vitro phenotypic ETV susceptibility was determined for virologic breakthrough isolates, and for HBV containing novel substitutions emerging during treatment. The results over 5 years of therapy showed that in nucleoside-naïve patients, the cumulative probability of genotypic ETVr and genotypic ETVr associated with virologic breakthrough was <em>1</em>.2% and 0.8%, respectively. In contrast, a reduced barrier to resistance was observed in LVD-refractory patients, as the LVD resistance substitutions, a partial requirement for ETVr, preexist, resulting in a 5-year cumulative probability of genotypic ETVr and genotypic ETVr associated with breakthrough of 5<em>1</em>% and 43%, respectively. Importantly, only four patients who achieved < 300 copies/<em>mL</em> HBV DNA subsequently developed ETVr.

CONCLUSIONS

Long-term monitoring showed low rates of resistance in nucleoside-naïve patients during 5 years of ETV therapy, corresponding with potent viral suppression and a high genetic barrier to resistance. These findings support ETV as a primary therapy that enables prolonged treatment with potent viral suppression and minimal resistance.

BACKGROUND

Chronic kidney disease (CKD) is common. Kidney disease severity can be classified by estimated glomerular filtration rate (GFR) and albuminuria, but more accurate information regarding risk for progression to kidney failure is required for clinical decisions about testing, treatment, and referral.

OBJECTIVE

To develop and validate predictive models for progression of CKD.

METHODS

Development and validation of prediction models using demographic, clinical, and laboratory data from 2 independent Canadian cohorts of patients with CKD stages 3 to 5 (estimated GFR, <em>1</em>0-59 <em>mL</em>/min/<em>1</em>.73 m(2)) who were referred to nephrologists between April <em>1</em>, 200<em>1</em>, and December 3<em>1</em>, 2008. Models were developed using Cox proportional hazards regression methods and evaluated using C statistics and integrated discrimination improvement for discrimination, calibration plots and Akaike Information Criterion for goodness of fit, and net reclassification improvement (NRI) at <em>1</em>, 3, and 5 years.

METHODS

Kidney failure, defined as need for dialysis or preemptive kidney transplantation.

RESULTS

The development and validation cohorts included 3449 patients (386 with kidney failure [<em>1</em><em>1</em>%]) and 4942 patients (<em>1</em><em>1</em>77 with kidney failure [24%]), respectively. The most accurate model included age, sex, estimated GFR, albuminuria, serum calcium, serum phosphate, serum bicarbonate, and serum albumin (C statistic, 0.9<em>1</em>7; 95% confidence interval [CI], 0.90<em>1</em>-0.933 in the development cohort and 0.84<em>1</em>; 95% CI, 0.825-0.857 in the validation cohort). In the validation cohort, this model was more accurate than a simpler model that included age, sex, estimated GFR, and albuminuria (integrated discrimination improvement, 3.2%; 95% CI, 2.4%-4.2%; calibration [Nam and D'Agostino χ(2) statistic, <em>1</em>9 vs 32]; and reclassification for CKD stage 3 [NRI, 8.0%; 95% CI, 2.<em>1</em>%-<em>1</em>3.9%] and for CKD stage 4 [NRI, 4.<em>1</em>%; 95% CI, -0.5% to 8.8%]).

CONCLUSIONS

A model using routinely obtained laboratory tests can accurately predict progression to kidney failure in patients with CKD stages 3 to 5.

Magnetic resonance (MR) tracking of magnetically labeled stem and progenitor cells is an emerging technology, leading to an urgent need for magnetic probes that can make cells highly magnetic during their normal expansion in culture. We have developed magnetodendrimers as a versatile class of magnetic tags that can efficiently label mammalian cells, including human neural stem cells (NSCs) and mesenchymal stem cells (MSCs), through a nonspecific membrane adsorption process with subsequent intracellular (non-nuclear) localization in endosomes. The superparamagnetic iron oxide nanocomposites have been optimized to exhibit superior magnetic properties and to induce sufficient MR cell contrast at incubated doses as low as <em>1</em> microg iron/<em>ml</em> culture medium. When containing between 9 and <em>1</em>4 pg iron/cell, labeled cells exhibit an ex vivo nuclear magnetic resonance (NMR) relaxation rate (<em>1</em>/T2) as high as 24-39 s-<em>1</em>/mM iron. Labeled cells are unaffected in their viability and proliferating capacity, and labeled human NSCs differentiate normally into neurons. Furthermore, we show here that NSC-derived (and LacZ-transfected), magnetically labeled oligodendroglial progenitors can be readily detected in vivo at least as long as six weeks after transplantation, with an excellent correlation between the obtained MR contrast and staining for beta-galactosidase expression. The availability of magnetodendrimers opens up the possibility of MR tracking of a wide variety of (stem) cell transplants.

OBJECTIVE

The authors hypothesized that pancreaticogastrostomy is safer than pancreaticojejunostomy after pancreaticoduodenectomy and less likely to be associated with a postoperative pancreatic fistula.

BACKGROUND

Pancreatic fistula is a leading cause of morbidity and mortality after pancreaticoduodenectomy, occurring in 10% to 20% of patients. Nonrandomized reports have suggested that pancreaticogastrostomy is less likely than pancreaticojejunostomy to be associated with postoperative complications.

METHODS

Between May 1993 and January 1995, the findings for 145 patients were analyzed in this prospective trial at The Johns Hopkins Hospital. After giving their appropriate preoperative informed consent, patients were randomly assigned to pancreaticogastrostomy or pancreaticojejunostomy after completion of the pancreaticoduodenal resection. All pancreatic anastomoses were performed in two layers without pancreatic duct stents and with closed suction drainage. Pancreatic fistula was defined as drainage of greater than 50 mL of amylase-rich fluid on or after postoperative day 10.

RESULTS

The pancreaticogastrostomy (n = 73) and pancreaticojejunostomy (n = 72) groups were comparable with regard to multiple parameters, including demographics, medical history, preoperative laboratory values, and intraoperative factors, such as operative time, blood transfusions, pancreatic texture, length of pancreatic remnant mobilized, and pancreatic duct diameter. The overall incidence of pancreatic fistula after pancreaticoduodenectomy was 11.7% (17/145). The incidence of pancreatic fistula was similar for the pancreaticogastrostomy (12.3%) and pancreaticojejunostomy (11.1%) groups. Pancreatic fistula was associated with a significant prolongation of postoperative hospital stay (36 +/- 5 vs. 15 +/- 1 days) (p < 0.001). Factors significantly increasing the risk of pancreatic fistula by univariate logistic regression analysis included ampullary or duodenal disease, soft pancreatic texture, longer operative time, greater intraoperative red blood cell transfusions, and lower surgical volume (p < 0.05). A multivariate logistic regression analysis revealed the factors most highly associated with pancreatic fistula to be lower surgical volume and ampullary or duodenal disease in the resected specimen.

CONCLUSIONS

Pancreatic fistula is a common complication after pancreaticoduodenectomy, with an incidence most strongly associated with surgical volume and underlying disease. These data do not support the hypothesis that pancreaticogastrostomy is safer than pancreaticojejunostomy or is associated with a lower incidence of pancreatic fistula.

Whole-cell assays were implemented to search for efflux pump inhibitors (EPIs) of the three multidrug resistance efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN) that contribute to fluoroquinolone resistance in clinical isolates of Pseudomonas aeruginosa. Secondary assays were developed to identify lead compounds with exquisite activities as inhibitors. A broad-spectrum EPI which is active against all three known Mex efflux pumps from P. aeruginosa and their close Escherichia coli efflux pump homolog (AcrAB-TolC) was discovered. When this compound, MC-207,<em>1</em><em>1</em>0, was used, the intrinsic resistance of P. aeruginosa to fluoroquinolones was decreased significantly (eightfold for levofloxacin). Acquired resistance due to the overexpression of efflux pumps was also decreased (32- to 64-fold reduction in the MIC of levofloxacin). Similarly, 32- to 64-fold reductions in MICs in the presence of MC-207,<em>1</em><em>1</em>0 were observed for strains with overexpressed efflux pumps and various target mutations that confer resistance to levofloxacin (e.g., gyrA and parC). We also compared the frequencies of emergence of levofloxacin-resistant variants in the wild-type strain at four times the MIC of levofloxacin (<em>1</em> microg/<em>ml</em>) when it was used either alone or in combination with EPI. In the case of levofloxacin alone, the frequency was approximately <em>1</em>0(-7) CFU/<em>ml</em>. In contrast, with an EPI, the frequency was below the level of detection ((<em>1</em>0(-<em>1</em><em>1</em>)). In summary, we have demonstrated that inhibition of efflux pumps (i) decreased the level of intrinsic resistance significantly, (ii) reversed acquired resistance, and (iii) resulted in a decreased frequency of emergence of P. aeruginosa strains that are highly resistant to fluoroquinolones.

Human recombinant tumor necrosis factor (rTNF) was found to act directly on cultured human vascular endothelium to induce a tissue factor-like procoagulant activity (PCA). After a 4-hr incubation in rTNF (<em>1</em>00 units/<em>ml</em>), serially passaged endothelial cells isolated from umbilical veins, saphenous veins, iliac arteries, and thoracic aortae demonstrated a dramatic increase (4- to <em>1</em>5-fold, 2<em>1</em> experiments) in total cellular PCA as measured with a one-stage clotting assay. rTNF-induced PCA was also expressed at the surface of intact viable endothelial monolayers. Induction of PCA by rTNF was concentration dependent (maximum, 500 units/<em>ml</em>), time dependent, reversible, and blocked by cycloheximide and actinomycin D, and it occurred without detectable endothelial cell damage. Actions of rTNF were compared with those of natural human interleukin <em>1</em> (IL-<em>1</em>) derived from stimulated monocytes and two distinct species of recombinant IL-<em>1</em>, each of which also induced endothelial PCA. The use of recombinant polypeptides and specific neutralizing antisera established the distinct natures of the mediators. The kinetics of the endothelial PCA responses to TNF and IL-<em>1</em> were similar, demonstrating a rapid rise to peak activity at approximately equal to 4 hr, and a decline toward basal levels by 24 hr. This characteristic decline in PCA after prolonged incubation with TNF or IL-<em>1</em> was accompanied by selective endothelial hyporesponsiveness to the initially stimulating monokine. Interestingly, the effects of TNF and IL-<em>1</em> were found to be additive even at apparent maximal doses of the individual monokines. Endothelial-directed actions of TNF, alone or in combination with other monokines, may be important in the initiation of coagulation and inflammatory responses in vivo.