While an elevated plasma concentration of HDLs is protective against the development of atherosclerosis and ensuing coronary heart disease (CHD), the mechanism of this protection is unknown. One early cellular event in atherogenesis is the adhesion of mononuclear leukocytes to the endothelium. This event is mediated principally by vascular cell adhesion molecule-<em>1</em> (VCAM-<em>1</em>) but also involves other molecules, such as intercellular adhesion molecule-<em>1</em> (ICAM-<em>1</em>) and E-selectin. We have investigated the effect of isolated plasma HDLs and reconstituted HDLs on the expression of these molecules by endothelial cells. We show that physiological concentrations of HDLs inhibit tumor necrosis factor-alpha (TNF-alpha) or interleukin-<em>1</em> (IL-<em>1</em>) induction of these leukocyte adhesion molecules in a concentration-dependent manner. Steady state mRNA levels of TNF-alpha-induced VCAM-<em>1</em> and E-selectin are significantly reduced by physiological concentrations of HDLs. An an HDL concentration of <em>1</em> mg/<em>mL</em> apolipoprotein A-I, the protein expressions of VCAM-<em>1</em>, ICAM-<em>1</em>, and E-selectin were inhibited by 89.6 +/- 0.4% (mean +/-SD, n=4), 64.8 +/- <em>1</em>.0%, and 79.2 +/- 0.4%, respectively. In contrast, HDLs have no effect on the expression of platelet endothelial cell adhesion molecule (PECAM) or on the expression of the p55 and p75 subunits of the TNF-alpha receptor. HDLs were effective when added from <em>1</em>6 hours before to 5 minutes after cytokine stimulation. HDLs had no effect on TNF-alpha-induced expression of ICAM-<em>1</em> by human foreskin fibroblasts, suggesting that the effect is cell-type restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

We conducted population-based surveillance for Candida bloodstream infections in Spain to determine its incidence, the extent of antifungal resistance, and risk factors for mortality. A case was defined as the first positive blood culture for any Candida spp. in a resident of Barcelona, from <em>1</em> January 2002 to 3<em>1</em> December 2003. We defined early mortality as occurring between days 3 to 7 after candidemia and late mortality as occurring between days 8 to 30. We detected 345 cases of candidemia, for an average annual incidence of 4.3 cases/<em>1</em>00,000 population, 0.53 cases/<em>1</em>,000 hospital discharges, and 0.73 cases/<em>1</em>0,000 patient-days. Outpatients comprised <em>1</em><em>1</em>% of the cases, and 89% had a central venous catheter (CVC) at diagnosis. Overall mortality was 44%. Candida albicans was the most frequent species (5<em>1</em>% of cases), followed by Candida parapsilosis (23%), Candida tropicalis (<em>1</em>0%), Candida glabrata (8%), Candida krusei (4%), and other species (3%). Twenty-four isolates (7%) had decreased susceptibility to fluconazole (MIC>> or = <em>1</em>6 microg/<em>ml</em>). On multivariable analysis, early death was independently associated with hematological malignancy (odds ratio [OR], 3.5; 95% confidence interval [CI], <em>1</em>.<em>1</em> to <em>1</em>0.4). Treatment with antifungals (OR, 0.05; 95% CI, 0.0<em>1</em> to 0.2) and removal of CVCs (OR, 0.3; 95% CI, 0.<em>1</em> to 0.9) were protective factors for early death. Receiving adequate treatment, defined as having CVCs removed and administration of an antifungal medication (OR, 0.2; 95% CI, 0.08 to 0.8), was associated with lower odds of late mortality; intubation (OR, 7.5; 95% CI, 2.6 to 2<em>1</em>.<em>1</em>) was associated with higher odds. The incidence of candidemia and prevalence of fluconazole resistance are similar to other European countries, indicating that routine antifungal susceptibility testing is not warranted. Antifungal medication and catheter removal are critical in preventing mortality.

Human neutrophil (PMN) attachment to human umbilical vein endothelial cells (HUVEC) was evaluated in vitro using two MAbs, R6-5-D6 and RR<em>1</em>/<em>1</em>, that recognize intercellular adhesion molecule-<em>1</em> (ICAM-<em>1</em>), and one MAb, TS<em>1</em>/<em>1</em>8, that recognizes CD<em>1</em>8. Pretreatment of the HUVEC with anti-ICAM-<em>1</em> MAbs produced greater than 50% inhibition of attachment to HUVEC, and IL-<em>1</em> (0.5 U/<em>ml</em>)- or lipopolysaccharide (LPS) (<em>1</em>0 ng/<em>ml</em>)-stimulated HUVEC, and greater than 99% inhibition of f-Met-Leu-Phe (0.5 nM) enhanced adherence. Anti-ICAM-<em>1</em> MAbs also inhibited by greater than 85% the transendothelial migration induced by 4-h IL-<em>1</em> (0.5 U/<em>ml</em>) and LPS (<em>1</em>0 ng/<em>ml</em>) activation of the HUVEC. That these effects involved a CD<em>1</em>8-dependent mechanism is supported by the following results: pretreatment of PMN with TS<em>1</em>/<em>1</em>8 produced the same degree of inhibition of attachment and migration as seen with R6-5-D6. In addition, the use of both MAbs together did not further increase the inhibition of cell attachment to stimulated HUVEC. The attachment of PMN from patients with CD<em>1</em>8 deficiency to stimulated HUVEC was not reduced by R6-5-D6, and both R6-5-D6 and TS<em>1</em>/<em>1</em>8 revealed the same time course for appearance and disappearance of an adherence component on stimulated HUVEC not blocked by either MAb. These results demonstrate that attachment and transendothelial migration of PMN in vitro depend substantially on both CD<em>1</em>8 on the PMN and ICAM-<em>1</em> on the endothelial cell.

A mainstay of strategies to prevent HIV-<em>1</em> transmission is to use antiretroviral therapy (ART) for pre-exposure prophylaxis (PrEP). Critical to the design and interpretation of PrEP prevention trials is the ability to make accurate pharmacological measurements of ART drugs in human genital and colorectal mucosal tissues, the principal route of HIV transmission. Here, we evaluated two drugs that are preferentially used for PrEP: tenofovir (TFV) disoproxil fumarate (TDF) and emtricitabine (FTC). A single oral dose of TDF/FTC (Truvada) was administered to <em>1</em>5 healthy individuals. Over the next <em>1</em>4 days, TFV and FTC were measured in blood plasma and genital secretions using a sensitive assay (lower level of quantification, 0.<em>1</em> ng/<em>ml</em>). The active intracellular phosphorylated metabolites of these drugs [TFV diphospate (TFV-DP) and FTC triphosphate (FTC-TP)] were measured in homogenates prepared from rectal, vaginal, and cervical tissues. TFV and FTC were detected in blood plasma <em>1</em>4 days after administration of a single dose. The area under the concentration-time curve from 24 hours to <em>1</em>4 days (AUC(<em>1</em>-<em>1</em>4d)) for FTC in genital secretions was 27-fold greater than in blood plasma, whereas the AUC(<em>1</em>-<em>1</em>4d) for TFV was only 2.5-fold greater in genital secretions than in blood plasma. In rectal tissue, TFV and TFV-DP concentrations were detectable for <em>1</em>4 days and were <em>1</em>00-fold higher than the concentrations in vaginal and cervical tissues. Vaginal and cervical tissue concentrations of FTC were <em>1</em>0- to <em>1</em>5-fold higher than in rectal tissue. Despite high concentrations of FTC in vaginal and cervical tissue, FTC-TP concentrations in all tissue types were detected for only 2 days after dose. The exposure to TFV, TFV-DP, FTC, and FTC-TP was wide ranging depending on the type of mucosal tissue. These results demonstrate the need for detailed pharmacological studies to improve the application of ART for PrEP to prevent transmission of HIV.

BACKGROUND

Exercise training in patients with chronic heart failure improves work capacity by enhancing endothelial function and skeletal muscle aerobic metabolism, but effects on central hemodynamic function are not well established.

OBJECTIVE

To evaluate the effects of exercise training on left ventricular (LV) function and hemodynamic response to exercise in patients with stable chronic heart failure.

METHODS

Prospective randomized trial conducted in <em>1</em>994-<em>1</em>999.

METHODS

University department of cardiology/outpatient clinic in Germany.

METHODS

Consecutive sample of 73 men aged 70 years or younger with chronic heart failure (with LV ejection fraction of approximately 0.27).

METHODS

Patients were randomly assigned to 2 weeks of in-hospital ergometer exercise for <em>1</em>0 minutes 4 to 6 times per day, followed by 6 months of home-based ergometer exercise training for 20 minutes per day at 70% of peak oxygen uptake (n=36) or to no intervention (control group; n=37).

METHODS

Ergospirometry with measurement of central hemodynamics by thermodilution at rest and during exercise; echocardiographic determination of LV diameters and volumes, at baseline and 6-month follow-up, for the exercise training vs control groups.

RESULTS

After 6 months, patients in the exercise training group had statistically significant improvements compared with controls in New York Heart Association functional class, maximal ventilation, exercise time, and exercise capacity as well as decreased resting heart rate and increased stroke volume at rest. In the exercise training group, an increase from baseline to 6-month follow-up was observed in mean (SD) resting LV ejection fraction (0.30 [0.08] vs 0.35 [0.09]; P=.003). Mean (SD) total peripheral resistance (TPR) during peak exercise was reduced by <em>1</em>57 (306) dyne/s/cm(-5) in the exercise training group vs an increase of 43 (<em>1</em>48) dyne/s/cm(-5) in the control group (P=.003), with a concomitant increase in mean (SD) stroke volume of <em>1</em>4 (22) <em>mL</em> vs <em>1</em> (<em>1</em>9) <em>mL</em> in the control group (P=.03). There was a small but significant reduction in mean (SD) LV end diastolic diameter of 4 (6) mm vs an increase of <em>1</em> (4) mm in the control group (P<.00<em>1</em>). Changes from baseline in resting TPR for both groups were correlated with changes in stroke volume (r=-0.76; P<.00<em>1</em>) and in LV end diastolic diameter (r=0.45; P<.00<em>1</em>).

CONCLUSIONS

In patients with stable chronic heart failure, exercise training is associated with reduction of peripheral resistance and results in small but significant improvements in stroke volume and reduction in cardiomegaly. JAMA. 2000.

Recently, we identified a novel crosstalk between insulin and G protein-coupled receptor (GPCR) signaling pathways in human pancreatic cancer cells. Insulin enhanced GPCR signaling through a rapamycin-sensitive mTOR-dependent pathway. Metformin, the most widely used drug in the treatment of type 2 diabetes, activates AMP kinase (AMPK), which negatively regulates mTOR. Here, we determined whether metformin disrupts the crosstalk between insulin receptor and GPCR signaling in pancreatic cancer cells. Treatment of human pancreatic cancer cells (PANC-<em>1</em>, MIAPaCa-2, and BxPC-3) with insulin (<em>1</em>0 ng/<em>mL</em>) for 5 minutes markedly enhanced the increase in intracellular [Ca(2+)] induced by GPCR agonists (e.g., neurotensin, bradykinin, and angiotensin II). Metformin pretreatment completely abrogated insulin-induced potentiation of Ca(2+) signaling but did not interfere with the effect of GPCR agonists alone. Insulin also enhanced GPCR agonist-induced growth, measured by DNA synthesis, and the number of cells cultured in adherent or nonadherent conditions. Low doses of metformin (0.<em>1</em>-0.5 mmol/L) blocked the stimulation of DNA synthesis, and the anchorage-dependent and anchorage-independent growth induced by insulin and GPCR agonists. Treatment with metformin induced striking and sustained increase in the phosphorylation of AMPK at Thr(<em>1</em>72) and a selective AMPK inhibitor (compound C, at 5 micromol/L) reversed the effects of metformin on [Ca(2+)](i) and DNA synthesis, indicating that metformin acts through AMPK activation. In view of these results, we tested whether metformin inhibits pancreatic cancer growth. Administration of metformin significantly decreased the growth of MIAPaCa-2 and PANC-<em>1</em> cells xenografted on the flank of nude mice. These results raise the possibility that metformin could be a potential candidate in novel treatment strategies for human pancreatic cancer.

Sustained virologic suppression is a primary goal of therapy for chronic hepatitis B (CHB). In study entecavir (ETV)-022, 48 weeks of entecavir 0.5 mg was superior to lamivudine for virologic suppression for hepatitis B e antigen (HBeAg)-positive CHB. A total of <em>1</em>83 entecavir-treated patients from ETV-022 subsequently enrolled in study ETV-90<em>1</em>. We present the results after up to 5 years (240 weeks) of continuous entecavir therapy. The entecavir long-term cohort consists of patients who received>>or=<em>1</em> year of entecavir 0.5 mg in ETV-022 and then entered ETV-90<em>1</em> with a treatment gap <or=35 days. In ETV-90<em>1</em> the entecavir dose was <em>1</em>.0 mg daily. For patients with samples available at Year 5, proportions with hepatitis B virus (HBV) DNA <300 copies/<em>mL</em>, normal alanine aminotransferase (ALT) levels, HBeAg loss, and HBeAg seroconversion were determined. In all, <em>1</em>46 patients met criteria for inclusion in the entecavir long-term cohort. At Year 5, 94% (88/94) had HBV DNA <300 copies/<em>mL</em> and 80% (78/98) had normal ALT levels. In addition to patients who achieved serologic responses during study ETV-022, 23% (33/<em>1</em>4<em>1</em>) achieved HBeAg seroconversion and <em>1</em>.4% (2/<em>1</em>45) lost hepatitis B surface antigen (HBsAg) during study ETV-90<em>1</em>. Through 5 years, entecavir resistance emerged in one patient. The safety profile of entecavir was consistent with previous reports.

CONCLUSIONS

Extended therapy with entecavir through 5 years maintained or increased rates of HBV DNA suppression and ALT normalization. Additional patients also achieved HBeAg loss and seroconversion. Entecavir provides sustained viral suppression with minimal resistance during long-term treatment of HBeAg-positive CHB.

Serum creatinine is not an ideal marker of renal function in patients with acute kidney injury (AKI). Previous studies demonstrated that urinary IL-<em>1</em>8 is increased in human AKI. Thus, whether urine IL-<em>1</em>8 is an early diagnostic marker of AKI was investigated. A nested case-control study was performed within the Acute Respiratory Distress Syndrome (ARDS) Network trial. AKI was defined as an increase in serum creatinine by at least 50% within the first 6 d of ARDS study enrollment. A total of 400 urine specimens that were collected on study days 0, <em>1</em>, and 3 of the ARDS trial were available from 52 case patients and 86 control patients. The data were analyzed in a cross-sectional manner and according to the time before development of AKI. The median urine IL-<em>1</em>8 levels were significantly different at 24 and 48 h before AKI in case patients as compared with control patients. On multivariable analysis, urine IL-<em>1</em>8 values predicted development of AKI 24 and 48 h later after adjustment for demographics, sepsis, Acute Physiologic Assessment and Chronic Health Evaluation (APACHE) III score, serum creatinine, and urine output. Urine IL-<em>1</em>8 levels of>><em>1</em>00 pg/<em>ml</em> are associated with increased odds of AKI of 6.5 (95% confidence interval 2.<em>1</em> to 20.4) in the next 24 h. On diagnostic performance testing, urine IL-<em>1</em>8 demonstrates an area under the receiver operating characteristic curve of 73% to predict AKI in the next 24 h. The urine IL-<em>1</em>8 values were also significantly different between survivors and nonsurvivors (P < 0.05), and on multivariable analysis, the urine IL-<em>1</em>8 value on day 0 is an independent predictor of mortality. Urinary IL-<em>1</em>8 levels can be used for the early diagnosis of AKI. Urine IL-<em>1</em>8 levels also predict the mortality of patients who have ARDS and are in the intensive care unit.

Historically, insulin resistance during pregnancy has been ascribed to increased production of placental hormones and cortisol. The purpose of this study was to test this hypothesis by correlating the longitudinal changes in insulin sensitivity during pregnancy with changes in placental hormones, cortisol, leptin, and tumor necrosis factor (TNF)-alpha. Insulin resistance was assessed in <em>1</em>5 women (5 with gestational diabetes mellitus [GDM] and <em>1</em>0 with normal glucose tolerance) using the euglycemic-hyperinsulinemic clamp procedure, before pregnancy (pregravid) and during early (<em>1</em>2-<em>1</em>4 weeks) and late (34-36 weeks) gestation. Body composition, plasma TNF-alpha, leptin, cortisol, and reproductive hormones (human chorionic gonadotropin, estradiol, progesterone, human placental lactogen, and prolactin) were measured in conjunction with the clamps. Placental TNF-alpha was measured in vitro using dually perfused human placental cotyledon from five additional subjects. Compared with pregravid, insulin resistance was evident during late pregnancy in all women (<em>1</em>2.4 +/- <em>1</em>.2 vs. 8.<em>1</em> +/- 0.8 <em>1</em>0(-2) mg. kg(-<em>1</em>) fat-free mass. min(-<em>1</em>). microU(-<em>1</em>). <em>ml</em>(-<em>1</em>)). TNF-alpha, leptin, cortisol, all reproductive hormones, and fat mass were increased in late pregnancy (P < 0.00<em>1</em>). In vitro, most of the placental TNF-alpha (94%) was released into the maternal circulation; 6% was released to the fetal side. During late pregnancy, TNF-alpha was inversely correlated with insulin sensitivity (r = -0.69, P < 0.006). Furthermore, among all of the hormonal changes measured in this study, the change in TNF-alpha from pregravid to late pregnancy was the only significant predictor of the change in insulin sensitivity (r = -0.60, P < 0.02). The placental reproductive hormones and cortisol did not correlate with insulin sensitivity in late pregnancy. Multivariate stepwise regression analysis revealed that TNF-alpha was the most significant independent predictor of insulin sensitivity (r = -0.67, P < 0.000<em>1</em>), even after adjustment for fat mass by covariance (r = 0.46, P < 0.0<em>1</em>). These observations challenge the view that the classical reproductive hormones are the primary mediators of change in insulin sensitivity during gestation and provide the basis for including TNF-alpha in a new paradigm to explain insulin resistance in pregnancy.

We previously reported encouraging results of down-staging of hepatocellular carcinoma (HCC) to meet conventional T2 criteria (one lesion 2-5 cm or two to three lesions <3 cm) for orthotopic liver transplantation (OLT) in 30 patients as a test of concept. In this ongoing prospective study, we analyzed longer-term outcome data on HCC down-staging in a larger cohort of 6<em>1</em> patients with tumor stage exceeding T2 criteria who were enrolled between June 2002 and January 2007. Eligibility criteria for down-staging included: (<em>1</em>) one lesion >5 cm and up to 8 cm; (2) two to three lesions with at least one lesion >3 cm and not exceeding 5 cm, with total tumor diameter up to 8 cm; or (3) four to five lesions with none >3 cm, with total tumor diameter up to 8 cm. A minimum observation period of 3 months after down-staging was required before OLT. Tumor down-staging was successful in 43 patients (70.5%). Thirty-five patients (57.4%) had received OLT, including two who had undergone live-donor liver transplantation. Treatment failure was observed in <em>1</em>8 patients (29.5%), primarily due to tumor progression. In the explant of 35 patients who underwent OLT, <em>1</em>3 had complete tumor necrosis, <em>1</em>7 met T2 criteria, and five exceeded T2 criteria. The Kaplan-Meier intention-to-treat survival at <em>1</em> and 4 years after down-staging were 87.5% and 69.3%, respectively. The <em>1</em>-year and 4-year posttransplantation survival rates were 96.2% and 92.<em>1</em>%, respectively. No patient had HCC recurrence after a median posttransplantation follow-up of 25 months. The only factor predicting treatment failure was pretreatment alpha-fetoprotein>><em>1</em>,000 ng/<em>mL</em>.

CONCLUSIONS

Successful down-staging of HCC can be achieved in the majority of carefully selected patients and is associated with excellent posttransplantation outcome.

Staphylococcal enterotoxins A-E (refs <em>1</em>-3), toxic shock toxin (TST-<em>1</em>) (ref. <em>1</em>), a product of Mycoplasma arthritidis and the <em>Mls</em> antigens provoke dramatic T-cell responses. All are extremely potent polyclonal mitogens stimulating a large proportion of both murine and human CD4+ and CD8+T cells although activity is tightly restricted by major histocompatibility complex (MHC) class II antigens. The murine T-cell response to staphylococcal enterotoxin B (SEB) has recently been shown to involve only those T cells expressing T-cell receptor V beta 3, 8.<em>1</em>, 8.2 and 8.3 domains, a situation which closely mimics the response to <em>Mls</em> antigens. This paper examines the initial events in SEA and SEB T-cell activation and shows that MHC restriction results from a direct high affinity binding by intact SEA and SEB to the same site on MHC class II HLA-DR antigens.

This study evaluated the efficacy and safety of donepezil in patients with mild to moderately severe Alzheimer's disease, and examined the relationships between plasma donepezil concentration, red blood cell acetylcholinesterase (AChE) activity and clinical response. The trial was of a multicenter, double-blind, parallel-group design and patients were randomised to once-daily treatment with either donepezil (<em>1</em>, 3 or 5 mg) or placebo. The <em>1</em>2-week double-blind phase was followed by a 2-week single-blind placebo washout. <em>1</em>6<em>1</em> patients (55-85 years of age) entered the study and <em>1</em>4<em>1</em> completed treatment. Patients treated with donepezil showed dose-related improvements in the Alzheimer's Disease Assessment Scale-cognitive subscale score (ADAS-cog) and in MMSF scores. The improvements in ADAS-cog were statistically significantly greater with donepezil 5 mg/day than with placebo. There was a 50% reduction in the percentage of patients showing clinical decline with donepezil at 5 mg/day (<em>1</em><em>1</em>%) relative to placebo (20%). In addition, a statistically significant correlation between plasma concentrations of donepezil and AChE inhibition was demonstrated. A plateau of inhibition (76-84%) was reached at plasma donepezil concentrations>> 50 ng/<em>ml</em>. The correlation between plasma drug concentrations and ADAS-cog (p = 0.0<em>1</em>4), MMSE (p = 0.023) and patient quality of life scores, assessed by the patient (p = 0.037) were also statistically significant, as was the correlation between AChE inhibition and change in ADAS-cog (p = 0.008). The incidence of treatment-emergent adverse events with all three dosages of donepezil (64-68%) was comparable to that observed with placebo (65%). Donepezil had no clinically significant effect on vital signs, haematology or clinical biochemistry tests. Importantly, donepezil was not associated with any hepatotoxicity, as observed with acridine-based cholinesterase inhibitors.

BACKGROUND

Molecular imaging of thrombus within fissures of vulnerable atherosclerotic plaques requires sensitive detection of a robust thrombus-specific contrast agent. In this study, we report the development and characterization of a novel ligand-targeted paramagnetic molecular imaging agent with high avidity for fibrin and the potential to sensitively detect active vulnerable plaques.

RESULTS

The nanoparticles were formulated with 2.5 to 50 mol% Gd-DTPA-BOA, which corresponds to >50 000 Gd(3+) atoms/particle. Paramagnetic nanoparticles were characterized in vitro and evaluated in vivo. In contradistinction to traditional blood-pool agents, T<em>1</em> relaxation rate as a function of paramagnetic nanoparticle number was increased monotonically with Gd-DTPA concentration from 0.<em>1</em>8 <em>mL</em>. s(-<em>1</em>). pmol(-<em>1</em>) (<em>1</em>0% Gd-DTPA nanoparticles) to 0.54 <em>mL</em>. s(-<em>1</em>). pmol(-<em>1</em>) for the 40 mol% Gd-DTPA formulations. Fibrin clots targeted in vitro with paramagnetic nanoparticles presented a highly detectable, homogeneous T<em>1</em>-weighted contrast enhancement that improved with increasing gadolinium level (0, 2.5, and 20 mol% Gd). Higher-resolution scans and scanning electron microscopy revealed that the nanoparticles were present as a thin layer over the clot surface. In vivo contrast enhancement under open-circulation conditions was assessed in dogs. The contrast-to-noise ratio between the targeted clot (20 mol% Gd-DTPA nanoparticles) and blood was approximately <em>1</em><em>1</em>8+/-2<em>1</em>, and that between the targeted clot and the control clot was <em>1</em>3<em>1</em>+/-37.

CONCLUSIONS

These results suggest that molecular imaging of fibrin-targeted paramagnetic nanoparticles can provide sensitive detection and localization of fibrin and may allow early, direct identification of vulnerable plaques, leading to early therapeutic decisions.

Organisms belonging to the Mycobacterium avium complex (MAC) are the most common bacterial pathogens in patients with AIDS but factors associated with the activation of cellular defense mechanisms against this atypical mycobacterium have not been defined. Peritoneal macrophages harvested from a chronic MAC infection in C57 black mice are able to kill approximately 86% of intracellular MAC in contrast to 0 to 20% killing by unstimulated human and mouse macrophages in vitro. The availability of human rTNF-alpha, rIFN-gamma, and rIL-2 permitted evaluation of the role of each of these lymphokines/monokines, alone or in combination, in activating macrophages in vitro to kill MAC. Human monocyte-derived macrophages were cultured in vitro, stimulated with rIL-2, rIFN-gamma, or rTNF, and then infected with MAC (serovars <em>1</em> and 8). Mouse peritoneal macrophages were harvested, cultured in vitro, and stimulated with rIFN-gamma. rTNF (<em>1</em>0(4) U/<em>ml</em>) was associated with a modest increase of intracellular killing of MAC (58 +/- 5%) even when utilized 24 or 48 h after macrophage infection or when administered for 5 consecutive days after infection (78.<em>1</em> +/- 4%). Both human and murine IFN-gamma were associated with increased intracellular growth of MAC (32 +/- 4% for murine and 38 +/- 3% for human macrophages). However, intracellular killing (53 +/- 6% compared with control) was observed after 6 days of treatment with IFN-gamma. This latter effect was fully blocked by anti-TNF antibody, whereas rIL-2 alone did not augment the intracellular killing of MAC by human macrophages. rTNF plus either rIFN-gamma or rIL-2 triggered significant increases in superoxide anion production, but subsequent MAC killing was no greater than with rTNF alone. Treatment of macrophages with <em>1</em>0 U/<em>ml</em> of rTNF followed by rIL-2 (200 U/<em>ml</em>) was associated with 68% of intracellular killing. TNF seems to be an important monokine, promoting activation of mycobactericidal mechanisms in human macrophages.

Human platelets contain a polypeptide growth factor that stimulates the proliferation of connective tissue cells. Purification of this platelet-derived growth factor (PDGF) was accomplished by heat (<em>1</em>00 degrees C) treatment of washed platelets and subsequent ion-exchange chromatography, gel filtration in <em>1</em> M acetic acid, isoelectric focusing, and preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis. PDGF has an isoelectric point of 9.8 and a molecular weight ranging from <em>1</em>3,000 to <em>1</em>6,000 as judged by gel filtration in <em>1</em> M acetic acid or analytical sodium dodecyl sulfate gel electrophoresis under reducing conditions. The specific activity of the purified PDGF is 20 million times greater than that found in unfractionated human serum. Purified PDGF stimulates replicative DNA synthesis and cell proliferation in quiescent density-arrested cultures of BALB/c 3T3 cells at concentrations of <em>1</em> ng/<em>ml</em> (0.<em>1</em> nM).

New broad and potent neutralizing HIV-<em>1</em> antibodies have recently been described that are largely dependent on the gp<em>1</em>20 N332 glycan for Env recognition. Members of the PGT<em>1</em>2<em>1</em> family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg <em>ml</em>(-<em>1</em>). Here, we show that three family members, PGT<em>1</em>2<em>1</em>, PGT<em>1</em>22 and PGT<em>1</em>23, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR<em>1</em>, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT<em>1</em>2<em>1</em> structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp<em>1</em>40 trimer (SOSIP.664) reveal that PGT<em>1</em>22 interacts with the gp<em>1</em>20 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT<em>1</em>28, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT<em>1</em>2<em>1</em> antibodies inhibit CD4 binding to gp<em>1</em>20 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT<em>1</em>2<em>1</em> antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V<em>1</em>/V2 complex biantennary glycan, are conformationally constrained.

Six Escherichia coli and <em>1</em>2 Klebsiella pneumoniae isolates from a single hospital expressed a common beta-lactamase with a pI of approximately 9.0 and were resistant to cefoxitin and cefotetan (MIC ranges, 64 to>> <em>1</em>28 and <em>1</em>6 to>> <em>1</em>28 micrograms/<em>ml</em>, respectively). Seventeen of the <em>1</em>8 strains produced multiple beta-lactamases. Most significantly, three K. pneumoniae strains were also resistant to imipenem (MICs, 8 to 32 micrograms/<em>ml</em>). Spectrophotometric beta-lactamase assays with purified enzyme indicated hydrolysis of cephamycins, in addition to cephaloridine and benzylpenicillin. The 4ene encoding the pI 9.0 beta-lactamase (designated ACT-<em>1</em> for AmpC type) was cloned and sequenced, which revealed an ampC-type beta-lactamase gene that originated from Enterobacter cloacae and that had 86% sequence homology to the P99 beta-lactamase and 94% homology to the partial sequence of MIR-<em>1</em>. Southern blotting revealed that the gene encoding ACT-<em>1</em> was on a large plasmid in some of the K. pneumoniae strains as well as on the chromosomes of all of the strains, suggesting that the gene is located on an easily mobilized element. Outer membrane protein profiles of the K. pneumoniae strains revealed that the three imipenem-resistant strains were lacking a major outer membrane protein of approximately 42 kDa which was present in the imipenem-susceptible strains. ACT-<em>1</em> is the first plasmid-mediated AmpC-type beta-lactamase derived from Enterobacter which has been completely sequenced. This work demonstrates that in addition to resistance to cephamycins, imipenem resistance can occur in K. pneumoniae when a high level of the ACT-<em>1</em> beta-lactamase is produced in combination with the loss of a major outer membrane protein.

This position paper on complementary feeding summarizes evidence for health effects of complementary foods. It focuses on healthy infants in Europe. After reviewing current knowledge and practices, we have formulated these conclusions: Exclusive or full breast-feeding for about 6 months is a desirable goal. Complementary feeding (ie, solid foods and liquids other than breast milk or infant formula and follow-on formula) should not be introduced before <em>1</em>7 weeks and not later than 26 weeks. There is no convincing scientific evidence that avoidance or delayed introduction of potentially allergenic foods, such as fish and eggs, reduces allergies, either in infants considered at increased risk for the development of allergy or in those not considered to be at increased risk. During the complementary feeding period, >90% of the iron requirements of a breast-fed infant must be met by complementary foods, which should provide sufficient bioavailable iron. Cow's milk is a poor source of iron and should not be used as the main drink before <em>1</em>2 months, although small volumes may be added to complementary foods. It is prudent to avoid both early (<4 months) and late >>or=7 months) introduction of gluten, and to introduce gluten gradually while the infant is still breast-fed, inasmuch as this may reduce the risk of celiac disease, type <em>1</em> diabetes mellitus, and wheat allergy. Infants and young children receiving a vegetarian diet should receive a sufficient amount ( approximately 500 <em>mL</em>) of breast milk or formula and dairy products. Infants and young children should not be fed a vegan diet.

BACKGROUND

Lung injury caused by a ventilator results from nonphysiologic lung stress (transpulmonary pressure) and strain (inflated volume to functional residual capacity ratio).

OBJECTIVE

To determine whether plateau pressure and tidal volume are adequate surrogates for stress and strain, and to quantify the stress to strain relationship in patients and control subjects.

METHODS

Nineteen postsurgical healthy patients (group <em>1</em>), <em>1</em><em>1</em> patients with medical diseases (group 2), 26 patients with acute lung injury (group 3), and 24 patients with acute respiratory distress syndrome (group 4) underwent a positive end-expiratory pressure (PEEP) trial (5 and <em>1</em>5 cm H2O) with 6, 8, <em>1</em>0, and <em>1</em>2 <em>ml</em>/kg tidal volume.

RESULTS

Plateau airway pressure, lung and chest wall elastances, and lung stress and strain significantly increased from groups <em>1</em> to 4 and with increasing PEEP and tidal volume. Within each group, a given applied airway pressure produced largely variable stress due to the variability of the lung elastance to respiratory system elastance ratio (range, 0.33-0.95). Analogously, for the same applied tidal volume, the strain variability within subgroups was remarkable, due to the functional residual capacity variability. Therefore, low or high tidal volume, such as 6 and <em>1</em>2 <em>ml</em>/kg, respectively, could produce similar stress and strain in a remarkable fraction of patients in each subgroup. In contrast, the stress to strain ratio-that is, specific lung elastance-was similar throughout the subgroups (<em>1</em>3.4 +/- 3.4, <em>1</em>2.6 +/- 3.0, <em>1</em>4.4 +/- 3.6, and <em>1</em>3.5 +/- 4.<em>1</em> cm H2O for groups <em>1</em> through 4, respectively; P = 0.58) and did not change with PEEP and tidal volume.

CONCLUSIONS

Plateau pressure and tidal volume are inadequate surrogates for lung stress and strain. Clinical trial registered with www.clinicaltrials.gov (NCT 00<em>1</em>43468).

BACKGROUND

Organochlorines such as DDT [2,2-bis(p-chlorophenyl)-<em>1</em>,<em>1</em>,<em>1</em>-trichloroethane] and PCBs (polychlorinated biphenyls), which have been used extensively as insecticides and as fluid insulators of electrical components, respectively, are known to be persistent environmental contaminants and animal carcinogens. These agents have been found in human tissue due to their inefficient metabolism and their solubility in lipids, which lead to lifelong sequestration in adipose tissue. Their association with human cancer occurrence, however, has been explored only marginally, with most studies having 20 or fewer cases.

OBJECTIVE

This blinded study was designed to determine whether exposure to PCBs and to DDE [<em>1</em>,<em>1</em>-dichloro-2,2-bis(p-chlorophenyl) ethylene], the major metabolite of DDT, is associated with breast cancer risk in women.

METHODS

We analyzed sera from the stored blood specimens of <em>1</em>4,290 participants enrolled between <em>1</em>985 and <em>1</em>99<em>1</em> in the New York University Women's Health Study, a prospective cohort study of hormones, diet, and cancer. Cohort members who developed breast cancer were included as case patients in our nested case-control study. DDE and PCBs were measured by gas chromatography in the sera of 58 women with a diagnosis of breast cancer <em>1</em>-6 months after they entered the cohort and in <em>1</em>7<em>1</em> matched control subjects from the same study population who did not develop cancer.

RESULTS

Mean levels of DDE and PCBs were higher for breast cancer case patients than for control subjects, but paired differences were statistically significant only for DDE (P = .03<em>1</em>). After adjustment for first-degree family history of breast cancer, lifetime lactation, and age at first full-term pregnancy, conditional logistic regression analysis showed a fourfold increase in relative risk of breast cancer for an elevation of serum DDE concentrations from 2.0 ng/mL (<em>1</em>0th percentile) to <em>1</em>9.<em>1</em> ng/mL (90th percentile). For PCBs, the relative risk for a change in serum levels from 3.9 ng/mL (<em>1</em>0th percentile) to <em>1</em>0.6 ng/mL (90th percentile) was less than twofold, a nonsignificant association that was further reduced after adjustment for DDE.

CONCLUSIONS

In this population of New York City women, breast cancer was strongly associated with DDE in serum but not with PCBs.

CONCLUSIONS

These findings suggest that environmental chemical contamination with organochlorine residues may be an important etiologic factor in breast cancer. Given the widespread dissemination of organochlorine insecticides in the environment and the food chain, the implications are far-reaching for public health intervention worldwide.

Interleukins (IL) -<em>1</em> beta and -<em>1</em> alpha and tumor necrosis factor (TNF-alpha) were measured by radioimmunoassay in plasma samples from 44 healthy individuals, <em>1</em>5 patients in septic shock, and 6 volunteers infused with endotoxin. Plasma IL-<em>1</em> alpha levels were low (40 pg/<em>ml</em>) or undetectable in all situations. In 67% of the healthy subjects, plasma IL-<em>1</em> beta levels were less than 70 pg/<em>ml</em>. Septic patients had higher plasma IL-<em>1</em> beta levels (<em>1</em>20 +/- <em>1</em>7 pg/<em>ml</em>, P = .00<em>1</em>); those of surviving patients were higher than those of patients who died (P = .05). Plasma TNF-alpha concentrations in septic individuals were elevated (<em>1</em><em>1</em>9 +/- 30 pg/<em>ml</em>) and correlated with severity of illness (r = .73, P = .003), but no correlation was observed between plasma IL-<em>1</em> beta and TNF-alpha concentrations in individual samples. Infusion of endotoxin caused a twofold elevation of IL-<em>1</em> beta, from a baseline of 35 +/- 5 pg/<em>ml</em> to a maximum of 69 +/- 27 pg/<em>ml</em> at <em>1</em>80 min (P less than .05). Peak TNF-alpha levels after endotoxin infusion were <em>1</em>5 times higher than IL-<em>1</em> beta levels, were attained more rapidly (90 min), and as with the septic patients, did not correlate with IL-<em>1</em> beta levels. These data support the concept that plasma IL-<em>1</em> beta and TNF-alpha concentrations are regulated independently and are associated with different clinical outcomes.

BACKGROUND

Vitamin D is essential for calcium metabolism as well as for fracture prevention, and a recent review suggested that the optimal serum 25(OH)D lies in the region of 50-80 nmol L-<em>1</em> (20-32 ng <em>mL</em>-<em>1</em>). A high prevalence of inadequacy has been reported in many studies but the prevalence of inadequacy amongst women with osteoporosis in different regions of the world has not been well characterized.

METHODS

A multinational study of <em>1</em>8 countries at various latitudes (range 64N-38S) was conducted in 2004 and 2005 to determine the average levels of serum 25(OH)D and the prevalence of vitamin D inadequacy. A total of 2606 postmenopausal women with osteoporosis (low bone mineral density, history of fragility fracture) seeking routine medical care were enrolled and serum 25(OH)D levels were measured at a single laboratory visit.

RESULTS

Mean serum 25(OH)D level was 26.8 ng <em>mL</em>-<em>1</em> (SE 0.3) and ranged from 7 to 243 ng <em>mL</em>-<em>1</em>. Regional mean values were highest in Latin America (29.6 ng <em>mL</em>-<em>1</em>, SE 0.6) and lowest in the Middle East (20.4 ng <em>mL</em>-<em>1</em>, SE 0.5). Overall, 64% of women had serum levels<30 ng <em>mL</em>-<em>1</em>. Serum parathyroid hormone reached a nadir at serum 25(OH)D levels>35 ng <em>mL</em>-<em>1</em>. In nonequatorial countries, women recruited during the winter months had somewhat lower serum 25(OH)D levels than those recruited during the summer months in some, but not all, countries.

CONCLUSIONS

Low levels of serum 25(OH)D are common amongst women with osteoporosis. The results underscore the value of assuring vitamin D adequacy in these women.

The sole human cathelicidin peptide, LL-37, has been demonstrated to protect animals against endotoxemia/sepsis. Low, physiological concentrations of LL-37 (< or =<em>1</em> microg/<em>ml</em>) were able to modulate inflammatory responses by inhibiting the release of the proinflammatory cytokine TNF-alpha in LPS-stimulated human monocytic cells. Microarray studies established a temporal transcriptional profile and identified differentially expressed genes in LPS-stimulated monocytes in the presence or absence of LL-37. LL-37 significantly inhibited the expression of specific proinflammatory genes up-regulated by NF-kappaB in the presence of LPS, including NFkappaB<em>1</em> (p<em>1</em>05/p50) and TNF-alpha-induced protein 2 (TNFAIP2). In contrast, LL-37 did not significantly inhibit LPS-induced genes that antagonize inflammation, such as TNF-alpha-induced protein 3 (TNFAIP3) and the NF-kappaB inhibitor, NFkappaBIA, or certain chemokine genes that are classically considered proinflammatory. Nuclear translocation, in LPS-treated cells, of the NF-kappaB subunits p50 and p65 was reduced>> or =50% in the presence of LL-37, demonstrating that the peptide altered gene expression in part by acting directly on the TLR-to-NF-kappaB pathway. LL-37 almost completely prevented the release of TNF-alpha and other cytokines by human PBMC following stimulation with LPS and other TLR2/4 and TLR9 agonists, but not with cytokines TNF-alpha or IL-<em>1</em>beta. Biochemical and inhibitor studies were consistent with a model whereby LL-37 modulated the inflammatory response to LPS/endotoxin and other agonists of TLR by a complex mechanism involving multiple points of intervention. We propose that the natural human host defense peptide LL-37 plays roles in the delicate balancing of inflammatory responses in homeostasis as well as in combating sepsis induced by certain TLR agonists.

BACKGROUND

Sustained suppression of the human immunodeficiency virus (HIV) type <em>1</em> RNA load with the use of highly active antiretroviral therapy (HAART) results in immunologic improvement, but it is not clear whether the CD4(+) cell count increases to normal levels or whether it reaches a less-than-normal plateau. We characterized the increase in the CD4(+) cell count in patients in clinical practice who maintained sustained viral suppression for up to 6 years.

METHODS

All patients were from the Johns Hopkins HIV Clinical Cohort, a longitudinal observational study of patients receiving primary HIV care in Baltimore, Maryland, who were observed for>><em>1</em> year while receiving HAART and who had sustained suppression of the HIV RNA load at <400 copies/mL. We analyzed annual change in the CD4(+) cell count for up to 6 years after the start of HAART, stratified by baseline CD4(+) cell counts of < or =200, 20<em>1</em>-350, >350 cells/microL, and we assessed the development of clinical events (death and new acquired immunodeficiency syndrome-defining illness) by Kaplan-Meier analysis.

RESULTS

A total of 655 patients were observed for a median of 46 months (range, <em>1</em>3-72 months). The median change from baseline to most recent CD4(+) cell count was +274 cells/microL, with 92% of patients having an increase in CD4(+) cell count. By 6 years, the median CD4(+) cell count was 493 cells/microL among patients with baseline CD4(+) cell counts < or =200 cells/microL, 508 cells/microL among those with baseline CD4(+) cell counts of 20<em>1</em>-350 cells/microL, and 829 cells/microL among those with baseline CD4(+) cell counts >350 cells/microL. In addition to baseline CD4(+) cell count, injection drug use and older age were associated with a lesser CD4(+) cell count response, and duration of therapy was associated with a greater CD4(+) cell count response.

CONCLUSIONS

Only patients with baseline CD4(+) cell counts >350 cells/microL returned to nearly normal CD4(+) cell counts after 6 years of follow-up. Significant increases were observed in all CD4(+) cell count strata during the first year, but there was a lower plateau CD4(+) cell count at lower baseline CD4(+) cell strata. These data suggest that waiting to start HAART at lower CD4(+) cell counts will result in the CD4(+) cell count not returning to normal levels.