The effect of minimally modified LDL (MM-LDL) on the ability of large vessel endothelial cells (EC) to interact with monocytes and neutrophils was examined. These LDL preparations, obtained by storage or by mild iron oxidation, were indistinguishable from native LDL to the LDL receptor and were not recognized by the scavenger receptor. Treatment of EC with as little as 0.<em>1</em>2 micrograms/<em>ml</em> MM-LDL caused a significant increase in the production of chemotactic factor for monocytes (sevenfold) and increased monocyte binding (three- to fivefold). Monocyte binding was maximal after 4 h of EC exposure to MM-LDL, persisted for 48 h, and was inhibited by cycloheximide. In contrast, neutrophil binding was not increased after <em>1</em>-24 h of exposure. Activity in the MM-LDL preparations was found primarily in the polar lipid fraction. MM-LDL was toxic for EC from one rabbit but not toxic for the cells from another rabbit or any human umbilical vein EC. The resistant cells became sensitive when incubated with lipoprotein in the presence of cycloheximide, whereas the sensitive strain became resistant when preincubated with sublethal concentrations of MM-LDL. We conclude that exposure of EC to sublethal levels of MM-LDL enhances monocyte endothelial interactions and induces resistance to the toxic effects of MM-LDL.

Adiponectin (ADPN), which is a secretory protein of adipose tissue, attenuates endothelial inflammatory responses in vitro. Among human subjects, plasma ADPN concentrations are reduced among patients with atherosclerotic complications but are substantially increased among patients with advanced renal failure. The clinical and biochemical correlates of plasma ADPN levels were investigated and the predictive power of ADPN levels with respect to survival rates and cardiovascular events was prospectively tested in a cohort of 227 hemodialysis patients, who were monitored for 3<em>1</em> +/- <em>1</em>3 mo. Plasma ADPN levels were 2.5 times higher (P < 0.000<em>1</em>) among dialysis patients (<em>1</em>5.0 +/- 7.7 microg/<em>ml</em>) than among healthy subjects (6.3 +/- 2.0 microg/<em>ml</em>), were independent of age, and were higher (P = 0.03) among women (<em>1</em>5.2 +/- 7.9 microg/<em>ml</em>) than among men (<em>1</em>4.0 +/- 7.4 microg/<em>ml</em>). For both genders, plasma ADPN levels were inversely related to body mass index values, plasma leptin levels, insulin levels, serum triglyceride levels, and homeostatic model assessment index values. Furthermore, plasma ADPN levels were directly related to HDL cholesterol levels and inversely related to von Willebrand factor levels. Plasma ADPN levels were lower (P < 0.05) among patients who experienced new cardiovascular events (<em>1</em>3.7 +/- 7.3 microg/<em>ml</em>) than among event-free patients (<em>1</em>5.8 +/- 7.8 microg/<em>ml</em>). There was a 3% risk reduction for each <em>1</em> microg/<em>ml</em> increase in plasma ADPN levels, and the relative risk of adverse cardiovascular events was <em>1</em>.56 times (95% confidence interval, <em>1</em>.<em>1</em>2 to <em>1</em>.99 times) higher among patients in the first ADPN tertile, compared with those in the third tertile. Plasma ADPN levels are an inverse predictor of cardiovascular outcomes among patients with end-stage renal disease. Furthermore, ADPN is related to several metabolic risk factors in a manner consistent with the hypothesis that this protein acts as a protective factor for the cardiovascular system.

OBJECTIVE

Transurethral resection of the prostate has for decades been the standard surgical therapy for lower urinary tract symptoms secondary to benign prostatic hyperplasia, the most common benign neoplasm in men. To generate a contemporary reference for evolving medical and minimally invasive therapies we analyzed complications and immediate outcomes of transurethral prostate resection in a statewide multicenter study.

METHODS

We prospectively evaluated <em>1</em>0,654 patients undergoing transurethral prostate resection in the state of Bavaria, Germany from January <em>1</em>, 2002 until December 3<em>1</em>, 2003. Case records containing 54 items concerning preoperative status, operation details, complications and immediate outcome, were recorded for each patient.

RESULTS

The mortality rate for transurethral prostate resection was 0.<em>1</em>0%. The cumulative short-term morbidity rate was <em>1</em><em>1</em>.<em>1</em>%. The most relevant complications were failure to void (5.8%), surgical revision (5.6%), significant urinary tract infection (3.6%), bleeding requiring transfusions (2.9%) and transurethral resection syndrome (<em>1</em>.4%). The resected tissue averaged 28.4 gm. Incidental carcinoma of the prostate was diagnosed by histological examination in 9.8% of patients. Urinary peak flow rate increased significantly to 2<em>1</em>.6 +/- 9.4 <em>ml</em> per second (baseline <em>1</em>0.4 +/- 6.8 <em>ml</em> per second, <em>1</em> tail p <0.000<em>1</em>), while post-void residual decreased to 3<em>1</em>.<em>1</em> +/- 73.0 <em>ml</em> (baseline <em>1</em>80.3 +/- 296.9 <em>ml</em>, <em>1</em>-tail p <0.000<em>1</em>).

CONCLUSIONS

In a large scale evaluation comprising 44 mostly nonacademic urological departments in Bavaria, unique real-world data for transurethral prostate resection were prospectively generated. This most contemporary information should be of use to potential patients and facilitate subsumption of emerging surgical and nonsurgical benign prostatic hyperplasia treatment options.

LPS stimulated human blood mononuclear leukocytes to produce a chemotactic factor for human neutrophils. The effect of LPS was dose-dependent; <em>1</em>0 micrograms/<em>ml</em> was optimal for production of chemotactic factor. Chemotactic activity was detected 3 hr after LPS stimulation, and reached its peak at <em>1</em>2 hr. No activity was detected in culture supernatants of unstimulated cells, provided LPS-free media were selected. Isoelectric point of the factor, determined by chromatofocusing, was approximately 8 to 8.5. Molecular weight was approximately <em>1</em>0 kilodaltons by Sephacryl S-200 gel filtration or by HPLC gel filtration on TSK-2000 and -3000 columns in succession. The gel filtration fractions were also assayed for IL <em>1</em> activity. The elution position of IL <em>1</em> activity corresponded to a m.w. of <em>1</em>8. There was no chemotactic activity in the IL <em>1</em> activity peak. Furthermore, highly purified natural Il <em>1</em> alpha and -beta and recombinant Il <em>1</em> alpha and -beta did not exhibit chemotactic activity for neutrophils in our assay. Among mononuclear leukocytes, the monocyte was the principal producer of neutrophil chemotactic factor. These results suggest that a chemotactic factor for neutrophils, different from IL <em>1</em>, is produced by LPS-stimulated blood monocytes.

BACKGROUND

Increased oxidative stress is important in the pathogenesis of chronic obstructive pulmonary disease (COPD). We postulated that treatment with the antioxidant N-acetylcysteine would reduce the rate of lung-function decline, reduce yearly exacerbation rate, and improve outcomes.

METHODS

In a randomised placebo-controlled study in 50 centres, 523 patients with COPD were randomly assigned to 600 mg daily N-acetylcysteine or placebo. Patients were followed for 3 years. Primary outcomes were yearly reduction in forced expiratory volume in <em>1</em> s (FEV<em>1</em>) and the number of exacerbations per year. Analysis was by intention to treat.

RESULTS

The yearly rate of decline in FEV<em>1</em> did not differ between patients assigned N-acetylcysteine and those assigned placebo (54 mL [SE 6] vs 47 mL [6]; difference in slope between groups 8 mL [9]; 95% CI -25 to <em>1</em>0). The number of exacerbations per year did not differ between groups (<em>1</em>.25 [SD <em>1</em>.35] vs <em>1</em>.29 [SD <em>1</em>.46]; hazard ratio 0.99 [95% CI 0.89-<em>1</em>.<em>1</em>0, p=0.85]). Subgroup analysis suggested that the exacerbation rate might be reduced with N acetylcysteine in patients not treated with inhaled corticosteroids and secondary analysis was suggestive of an effect on hyperinflation.

CONCLUSIONS

N-acetylcysteine is ineffective at prevention of deterioration in lung function and prevention of exacerbations in patients with COPD.

Two different cell populations, high- (MARC-<em>1</em>45) and low-permissive cell clones (L-<em>1</em>) to porcine reproductive and respiratory syndrome (PRRS) virus, were derived from MA-<em>1</em>04 cell line (parent cell: P) by cell cloning. Maximum virus yields in MARC-<em>1</em>45, P, and L-<em>1</em> cell clones were <em>1</em>0(8.5), <em>1</em>0(3.5), and <em>1</em>0(2.5) tissue culture infective dose 50 (TCID50)/0.<em>1</em> <em>ml</em>, respectively. The MARC-<em>1</em>45 cell clone supported replication of all <em>1</em><em>1</em> different porcine reproductive and respiratory syndrome virus isolates that were tested. These results indicated that the MARC-<em>1</em>45 cells will be useful for PRRS virus replication.

BACKGROUND

Recent literature in mechanical ventilation includes strong evidence from randomized trials. Little information is available regarding the influence of these trials on usual clinical practice.

OBJECTIVE

To describe current mechanical ventilation practices and to assess the influence of interval randomized trials when compared with findings from a <em>1</em>998 cohort.

METHODS

A prospective international observational cohort study, with a nested comparative study performed in 349 intensive care units in 23 countries. We enrolled 4,968 consecutive patients receiving mechanical ventilation over a <em>1</em>-month period. We recorded demographics and daily data related to mechanical ventilation for the duration of ventilation. We systematically reviewed the literature and developed <em>1</em><em>1</em> practice-change hypotheses for the comparative cohort study before seeing these results. In assessing practice changes, we only compared data from the <em>1</em>07 intensive care units (<em>1</em>,675 patients) that also participated in the <em>1</em>998 cohort (<em>1</em>,383 patients).

RESULTS

In 2004 compared with <em>1</em>998, the use of noninvasive ventilation increased (<em>1</em><em>1</em>.<em>1</em> vs. 4.4%, P < 0.00<em>1</em>). Among patients with acute respiratory distress syndrome, tidal volumes decreased (7.4 vs. 9.<em>1</em> ml/kg, P < 0.00<em>1</em>) and positive end-expiratory pressure levels increased slightly (8.7 vs. 7.7 cm H(2)O, P = 0.02). More patients were successfully extubated after their first attempt of spontaneous breathing (77 vs. 62%, P < 0.00<em>1</em>). Use of synchronized intermittent mandatory ventilation fell dramatically (<em>1</em>.6 vs. <em>1</em><em>1</em>%, P < 0.00<em>1</em>). Observations confirmed <em>1</em>0 of our <em>1</em><em>1</em> practice-change hypotheses.

CONCLUSIONS

The strong concordance of predicted and observed practice changes suggests that randomized trial results have advanced mechanical ventilation practices internationally.

Treatment of human HL-60 or KG<em>1</em>A leukemia cells with the topoisomerase II inhibitor etoposide resulted in extensive DNA degradation. When DNA integrity was analyzed by agarose gel electrophoresis, a nucleosomal ladder became evident <em>1</em>.5-2 h after addition of etoposide to cells, increased in intensity over 6 h, and persisted at 24 h. Six h after addition of the drug, 94 +/- 4% of the cells excluded trypan blue even though as much as 90% of the DNA had been degraded to oligosomal fragments. Exposure of cells to <em>1</em>0 micrograms/<em>ml</em> (<em>1</em>7 microM) etoposide for as little as 45 min was sufficient to induce this DNA damage 4 h later. Preincubation with dinitrophenol abolished the effect of etoposide, suggesting that an energy-requiring step occurred prior to or during the endonucleolytic cleavage. In contrast, the effect of etoposide was not prevented by preincubation of HL-60 cells with the RNA synthesis inhibitor 5,6-dichloro-<em>1</em>-beta-ribofuranosylbenzimidazole or the protein synthesis inhibitors cycloheximide or puromycin. On the contrary, high concentrations of 5,6-dichloro-<em>1</em>-beta-ribofuranosylbenzimidazole, cycloheximide, or puromycin by themselves induced the same endonucleolytic cleavage, as did a variety of diverse cytotoxic agents, including camptothecin (0.<em>1</em> microM), colcemid (0.<em>1</em> microgram/<em>ml</em>), cis-platinum (20 microM), methotrexate (<em>1</em> microM), and <em>1</em>-beta-D-arabinofuranosylcytosine (3 microM). These results suggest that endonucleolytic DNA damage by a preexisting cellular enzyme occurs soon after treatment of HL-60 cells with any of a variety of cytotoxic agents. The observation that a variety of nuclear proteins [including poly(ADP-ribose) polymerase, lamin B, topoisomerase I, topoisomerase II, and histone H<em>1</em>] are degraded concomitant with the DNA fragmentation calls into question the selectivity of the degradative process for DNA. The implications of these results for (a) current theories which focus upon endonucleolytic damage of DNA as a critical early event during cell death, and (b) use of topoisomerase-directed drugs to map topoisomerase-binding sites in active chromatin are discussed.

Interleukin 3 (IL 3) was initially defined as a factor in conditioned media from concanavalin A-stimulated lymphocytes (Con A CM) that induces the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in cultures of nu/nu splenic lymphocytes. To determine the spectrum of additional "biologic" activities, IL 3 was purified to homogeneity and its properties were assessed. The protein preparation was judged to be homogeneous IL 3 by the following criteria: <em>1</em>) elution of a peak of IL 3 with a constant specific activity in the last step of purification, 2) presence of a single protein by SDS-PAGE analysis, 3) receptor-binding activity against IL 3-dependent cell lines, 4) a specific activity of congruent to 0.2 ng/<em>ml</em> required for 50% of maximal biologic activity, and 5) the presence of a single amino terminal sequence. With the use of this preparation of IL 3, the dose-response curves for 20 alpha SDH induction were identical or similar to the dose-response curves for the activities of <em>1</em>) WEHI-3 growth factor, 2) mast cell growth factor, 3) P cell-stimulating factor, and 4) histamine-producing cell-stimulating factor. In addition, homogeneous IL 3 had colony-stimulating factor activity, although only approximately 2% of the total CSF activity found in Con A CM was associated with IL 3. The major peak of CSF activity could be resolved from IL 3 by DEAE column chromatography and lacked many of the biologic activities associated with IL 3.

BACKGROUND

Despite advances in clinical management, there are currently no reliable diagnostic and therapeutic targets for acute respiratory distress syndrome (ARDS). The inflammasome/caspase-<em>1</em> pathway regulates the maturation and secretion of proinflammatory cytokines (e.g., IL-<em>1</em>8). IL-<em>1</em>8 is associated with injury in animal models of systemic inflammation.

OBJECTIVE

We sought to determine the contribution of the inflammasome pathway in experimental acute lung injury and human ARDS.

METHODS

We performed comprehensive gene expression profiling on peripheral blood from patients with critical illness. Gene expression changes were assessed using real-time polymerase chain reaction, and IL-<em>1</em>8 levels were measured in the plasma of the critically ill patients. Wild-type mice or mice genetically deficient in IL-<em>1</em>8 or caspase-<em>1</em> were mechanically ventilated using moderate tidal volume (<em>1</em>2 ml/kg). Lung injury parameters were assessed in lung tissue, serum, and bronchoalveolar lavage fluid.

RESULTS

In mice, mechanical ventilation enhanced IL-<em>1</em>8 levels in the lung, serum, and bronchoalveolar lavage fluid. IL-<em>1</em>8-neutralizing antibody treatment, or genetic deletion of IL-<em>1</em>8 or caspase-<em>1</em>, reduced lung injury in response to mechanical ventilation. In human patients with ARDS, inflammasome-related mRNA transcripts (CASP<em>1</em>, IL<em>1</em>B, and IL<em>1</em>8) were increased in peripheral blood. In samples from four clinical centers, IL-<em>1</em>8 was elevated in the plasma of patients with ARDS (sepsis or trauma-induced ARDS) and served as a novel biomarker of intensive care unit morbidity and mortality.

CONCLUSIONS

The inflammasome pathway and its downstream cytokines play critical roles in ARDS development.

OBJECTIVE

Trastuzumab-DM<em>1</em> (T-DM<em>1</em>) is an antibody-drug conjugate that uses trastuzumab to specifically deliver the maytansinoid antimicrotubule agent DM<em>1</em> to HER2-positive cells. This first-in-human study of T-DM<em>1</em> evaluated safety, pharmacokinetics, and preliminary activity of T-DM<em>1</em> in patients with advanced HER2-positive breast cancer.

METHODS

Successive cohorts of patients who had progressed on trastuzumab-based therapy received escalating doses of T-DM<em>1</em>. Outcomes were assessed by standard solid-tumor phase I methods.

RESULTS

Twenty-four patients who had received a median of four prior chemotherapeutic agents for metastatic disease received T-DM<em>1</em> at 0.3 mg/kg to 4.8 mg/kg on an every-3-weeks schedule. Transient thrombocytopenia was dose-limiting at 4.8 mg/kg; the maximum-tolerated dose (MTD) was 3.6 mg/kg. The half-life of T-DM<em>1</em> at the MTD was 3.5 days, with peak DM<em>1</em> levels < <em>1</em>0 ng/<em>mL</em>. Clearance at doses < <em>1</em>.2 mg/kg was faster than at higher doses. Common drug-related adverse events (AEs) included grade < or = 2 thrombocytopenia, elevated transaminases, fatigue, nausea, and anemia. No grade>> <em>1</em> nausea, vomiting, alopecia, or neuropathy events and no cardiac effects requiring dose modification were reported. The clinical benefit rate (objective response plus stable disease at 6 months) among <em>1</em>5 patients treated at the MTD was 73%, including five objective responses. The confirmed response rate in patients with measurable disease at the MTD (n = 9) was 44%.

CONCLUSIONS

At the MTD of 3.6 mg/kg every 3 weeks, T-DM<em>1</em> was associated with mild, reversible toxicity and substantial clinical activity in a heavily pretreated population. Phase II and III trials in patients with advanced HER2-positive breast cancer are under way.

OBJECTIVE

Cell-mediated immunity is a feature of Crohn's disease (CD). The heterodimer interleukin (IL)-<em>1</em>2, produced by phagocytes, induces T-cell cytokines, primarily interferon (IFN)-gamma. This study examined whether CD lamina propria mononuclear cells (LPMCs) express and release bioactive IL-<em>1</em>2.

METHODS

LPMCs were isolated from <em>1</em>3 patients with CD, 9 with ulcerative colitis (UC), and <em>1</em>3 controls. Messenger RNA for p40 and p35 IL-<em>1</em>2 subunits was evaluated by reverse-transcription polymerase chain reaction. IL-<em>1</em>2 was measured by enzyme-linked immunosorbent assay in LPMC culture supernatants. The INF-gamma-inducing effect of unstimulated LPMC supernatants was evaluated.

RESULTS

Messenger RNA for both IL-<em>1</em>2 subunits was detected in LPMCs of <em>1</em><em>1</em> of <em>1</em>3 patients with CD, <em>1</em> of 9 patients with UC, and <em>1</em> of <em>1</em>3 controls (P < 0.00<em>1</em>). IL-<em>1</em>2 was measured (<em>1</em>0.5 +/- 2 pg/<em>mL</em> at 24 hours) in unstimulated CD LPMCs and was enhanced by pokeweed mitogen, lipopolysaccharide, and staphylococcal enterotoxin B. No IL-<em>1</em>2 was detectable in 8 of 9 patients with UC and <em>1</em>2 of <em>1</em>3 control-unstimulated LPMCs. IL-<em>1</em>2 induced by pokeweed mitogen and staphylococcal enterotoxin B in UC was lower than in CD and did not differ from controls. An IFN-gamma-inducing effect was restricted to unstimulated CD LPMC supernatants and was inhibited by an anti-IL-<em>1</em>2 antibody in a dose-dependent fashion.

CONCLUSIONS

IL-<em>1</em>2 transcripts are expressed in CD intestinal tissues. CD LPMCs are up-regulated in their capability of releasing bioactive IL-<em>1</em>2. Expression and release of bioactive IL-<em>1</em>2 seem to differentiate CD from UC.

The oscillatory properties of the membrane potential in inferior olivary neurones were studied in guinea-pig brain-stem slices maintained in vitro. Intracellular double-ramp current injection at frequencies of <em>1</em>-20 Hz revealed that inferior olivary neurones tend to fire at two preferred frequencies: 3-6 Hz when the cells were actively depolarized (resting potential less than -50 mV), and 9-<em>1</em>2 Hz when they were actively hyperpolarized (resting potential more than -75 mV). In <em>1</em>0% of the experiments spontaneous subthreshold oscillations of the membrane potential were observed. These oscillations, which resembled sinusoidal wave forms and had a frequency of 4-6 Hz and an amplitude of 5-<em>1</em>0 mV, occurred synchronously in all cells tested within the slice. These oscillations persisted in the presence of <em>1</em>0(-4) M-tetrodotoxin and were blocked by Ca2+ conductance blockers or by the removal of Ca2+ from the bathing solution. The oscillations were affected by gross extracellular stimulation of the slice but not by intracellular activation of any given neurone. The data indicate that these oscillations reflect the properties of neuronal ensembles comprised of a large number of coupled elements. Similar ensemble oscillation could be induced, in most experiments, by adding harmaline (0.<em>1</em> mg/<em>ml</em>) and serotonin (<em>1</em>0(-4) M) to the bath and could be blocked by bath addition of noradrenaline. Harmaline was found to increase cell excitability by hyperpolarizing the neurones and shifting the inactivation curve for the somatic Ca2+ spike to a more positive membrane potential level. The role inferior olivary oscillations play in the organization of motor coordination is discussed.

BACKGROUND

We previously estimated the prevalence of chronic kidney disease (CKD) stages 3-5 at <em>1</em>9.<em>1</em> million based on data from the Japanese annual health check program for 2000-2004 using the Modification of Diet in Renal Disease (MDRD) equation multiplied by the coefficient 0.88<em>1</em> for the Japanese population. However, this equation underestimates the GFR, particularly for glomerular filtration rates (GFRs) of over 60 <em>ml</em>/min/<em>1</em>.73 m(2). We did not classify the participants as CKD stages <em>1</em> and 2 because we did not obtain proteinuria data for all of the participants. We re-estimated the prevalence of CKD by measuring proteinuria using a dipstick test and by calculating the GFR using a new equation that estimates GFR based on data from the Japanese annual health check program in 2005.

METHODS

Data were obtained for 574,024 (male 240,594, female 333,430) participants over 20 years old taken from the general adult population, who were from <em>1</em><em>1</em> different prefectures in Japan (Hokkaido, Yamagata, Fukushima, Tochigi, Ibaraki, Tokyo, Kanazawa, Osaka, Fukuoka, Miyazaki and Okinawa) and took part in the annual health check program in 2005. The glomerular filtration rate (GFR) of each participant was computed from the serum creatinine value using a new equation: GFR (<em>ml</em>/min/<em>1</em>.73 m(2)) = <em>1</em>94 x Age(-0.287) x S-Cr(-<em>1</em>.094) (if female x 0.739). The CKD population nationwide was calculated using census data from 2005. We also recalculated the prevalence of CKD in Japan assuming that the age composition of the population was same as that in the USA.

RESULTS

The prevalence of CKD stages <em>1</em>, 2, 3, and 4 + 5 were 0.6, <em>1</em>.7, <em>1</em>0.4 and 0.2% in the study population, which resulted in predictions of 0.6, <em>1</em>.7, <em>1</em>0.7 and 0.2 million patients, respectively, nationwide. The prevalence of low GFR was significantly higher in the hypertensive and proteinuric populations than it was in the populations without proteinuria or hypertension. The prevalence rate of CKD in Japan was similar to that in the USA when the Japanese general population was age adjusted to the US 2005 population estimate.

CONCLUSIONS

About <em>1</em>3% of the Japanese adult population-approximately <em>1</em>3.3 million people-were predicted to have CKD in 2005.

Transient antiretroviral treatment with tenofovir, (R)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than <em>1</em> year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8(+) lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8(+) cells and returned to predepletion levels (as low as (<em>1</em>00 copy Eq/<em>ml</em>) as circulating CD8(+) cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.

BACKGROUND

Plasma human immunodeficiency virus (HIV) RNA level predicts HIV disease progression, but the extent to which it explains the variability in rate of CD4 cell depletion is poorly characterized.

OBJECTIVE

To estimate the proportion of variability in rate of CD4 cell loss predicted by presenting plasma HIV RNA levels in untreated HIV-infected persons.

METHODS

Repeated-measures analyses of 2 multicenter cohorts, comprising observations beginning on May <em>1</em>2, <em>1</em>984, and ending on August 26, 2004. Analyses were conducted between August 2004 and March 2006.

METHODS

Two cohorts of HIV-infected persons: patients followed up at 4 US teaching medical institutions or participating in either the Research in Access to Care for the Homeless Cohort (REACH) or the San Francisco Men's Health Study (SFMHS) cohorts and participants in the Multicenter AIDS Cohort Study (MACS) cohort.

METHODS

Antiretroviral treatment-naive, chronically HIV-infected persons (n = <em>1</em>289 and n = <em>1</em>5<em>1</em>2 for each of the 2 cohorts) untreated during the observation period >> or =6 months) and with at least <em>1</em> HIV RNA level and 2 CD4 cell counts available. Approximately 35% were nonwhite, and 35% had risk factors other than male-to-male sexual contact.

METHODS

The extent to which presenting plasma HIV RNA level could explain the rate of model-derived yearly CD4 cell loss, as estimated by the coefficient of determination (R2).

RESULTS

In both cohorts, higher presenting HIV RNA levels were associated with greater subsequent CD4 cell decline. In the study cohort, median model-estimated CD4 cell decrease among participants with HIV RNA levels of 500 or less, 50<em>1</em> to 2000, 200<em>1</em> to <em>1</em>0,000, <em>1</em>0,00<em>1</em> to 40,000, and more than 40,000 copies/mL were 20, 39, 48, 56, and 78 cells/microL, respectively. Despite this trend across broad categories of HIV RNA levels, only a small proportion of CD4 cell loss variability (4%-6%) could be explained by presenting plasma HIV RNA level. Analyses using multiple HIV RNA measurements or restricting to participants with high HIV RNA levels improved this correlation minimally (R2, 0.09), and measurement error was estimated to attenuate these associations only marginally (deattenuated R2 in the 2 cohorts, 0.05 and 0.08, respectively).

CONCLUSIONS

Presenting HIV RNA level predicts the rate of CD4 cell decline only minimally in untreated persons. Other factors, as yet undefined, likely drive CD4 cell losses in HIV infection. These findings have implications for treatment decisions in HIV infection and for understanding the pathogenesis of progressive immune deficiency.

Chitosan-DNA nanoparticles were prepared using a complex coacervation process. The important parameters for the nanoparticle synthesis were investigated, including the concentrations of DNA, chitosan and sodium sulfate, temperature of the solutions, pH of the buffer, and molecular weights of chitosan and DNA. At an amino group to phosphate group ratio (N/P ratio) between 3 and 8 and a chitosan concentration of <em>1</em>00 microg/<em>ml</em>, the size of particles was optimized to approximately <em>1</em>00--250 nm with a narrow distribution, with a composition of 35.6 and 64.4% by weight for DNA and chitosan, respectively. The surface charge of these particles was slightly positive with a zeta potential of +<em>1</em>2 to +<em>1</em>8 mV at pH lower than 6.0, and became nearly neutral at pH 7.2. The chitosan-DNA nanoparticles could partially protect the encapsulated plasmid DNA from nuclease degradation as shown by electrophoretic mobility analysis. The transfection efficiency of chitosan-DNA nanoparticles was cell-type dependent. Typically, it was three to four orders of magnitude, in relative light units, higher than background level in HEK293 cells, and two to ten times lower than that achieved by LipofectAMINE-DNA complexes. The presence of <em>1</em>0% fetal bovine serum did not interfere with their transfection ability. Chloroquine could be co-encapsulated in the nanoparticles at 5.2%, but with negligible enhancement effect despite the fact that chitosan only showed limited buffering capacity compared with PEI. The present study also developed three different schemes to conjugate transferrin or KNOB protein to the nanoparticle surface. The transferrin conjugation only yielded a maximum of four-fold increase in their transfection efficiency in HEK293 cells and HeLa cells, whereas KNOB conjugated nanoparticles could improve gene expression level in HeLa cells by <em>1</em>30-fold. Conjugation of PEG on the nanoparticles allowed lyophilization without aggregation, and without loss of bioactivity for at least <em>1</em> month in storage. The clearance of the PEGylated nanoparticles in mice following intravenous administration was slower than unmodified nanoparticles at <em>1</em>5 min, and with higher depositions in kidney and liver. However, no difference was observed at the <em>1</em>-h time point.

Recently, the first Neisseria gonorrhoeae strain (H04<em>1</em>) highly resistant to the expanded-spectrum cephalosporins (ESCs) ceftriaxone and cefixime, which are the last remaining options for first-line gonorrhea treatment, was isolated in Japan. Here, we confirm and characterize a second strain (F89) with high-level cefixime and ceftriaxone resistance which was isolated in France and most likely caused a treatment failure with cefixime. F89 was examined using six species-confirmatory tests, antibiograms (33 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of known gonococcal resistance determinants (penA, mtrR, penB, ponA, and pilQ). F89 was assigned to MLST sequence type <em>1</em>90<em>1</em> (ST<em>1</em>90<em>1</em>) and NG-MAST ST<em>1</em>407, which is a successful gonococcal clone that has spread globally. F89 has high-level resistance to cefixime (MIC = 4 μg/<em>ml</em>) and ceftriaxone (MIC = <em>1</em> to 2 μg/<em>ml</em>) and resistance to most other antimicrobials examined. A novel penA mosaic allele (penA-CI), which was penA-XXXIV with an additional A50<em>1</em>P alteration in penicillin-binding protein 2, was the primary determinant for high-level ESC resistance, as determined by transformation into a set of recipient strains. N. gonorrhoeae appears to be emerging as a superbug, and in certain circumstances and settings, gonorrhea may become untreatable. Investigations of the biological fitness and enhanced understanding and monitoring of the ESC-resistant clones and their international transmission are required. Enhanced disease control activities, antimicrobial resistance control and surveillance worldwide, and public health response plans for global (and national) perspectives are also crucial. Nevertheless, new treatment strategies and/or drugs and, ideally, a vaccine are essential to develop for efficacious gonorrhea management.

Interactions of polycationic polymers with supported <em>1</em>,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and live cell membranes (KB and Rat2) have been investigated using atomic force microscopy (AFM), cytosolic enzyme assays, confocal laser scanning microscopy (CLSM), and a fluorescence-activated cell sorter (FACS). Polycationic polymers poly-L-lysine (PLL), polyethylenimine (PEI), and diethylaminoethyl-dextran (DEAE-DEX) and sphere-like poly(amidoamine) (PAMAM) dendrimers are employed because of their importance for gene and drug delivery. AFM studies indicate that all the polycationic polymers cause the formation and/or expansion of preexisting defects in supported DMPC bilayers in the concentration range of <em>1</em>-3 microg/<em>mL</em>. By way of contrast, hydroxyl-containing neutral linear poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) do not induce hole formation or expand the size of preexisting defects in the same concentration range. All polymers tested are not toxic to KB or Rat2 cells up to a <em>1</em>2 microg/<em>mL</em> concentration (XTT assay). In the concentration range of 6-<em>1</em>2 microg/<em>mL</em>, however, significant amounts of the cytosolic enzymes lactate dehydrogenase (LDH) and luciferase (LUC) are released. PEI, which possesses the greatest density of charged groups on its chain, shows the most dramatic increase in membrane permeability. In addition, treatment with polycationic polymers allows the small dye molecules propidium idodide (PI) and fluorescein (FITC) to diffuse in and out of the cells. CLSM images also show internalization of PLL labeled with FITC dye. In contrast, controls of membrane permeability using the neutral linear polymers PEG and PVA show dramatically less LDH and LUC leakage and no enhanced dye diffusion. Taken together, these data are consistent with the hypothesis that polycationic polymers induce the formation of transient, nanoscale holes in living cells and that these holes allow a greatly enhanced exchange of materials across the cell membrane.

BACKGROUND

The MRKAd5 HIV-<em>1</em> gag/pol/nef subtype B vaccine was designed to elicit T-cell-mediated immune responses capable of providing complete or partial protection from HIV-<em>1</em> infection or a decrease in viral load after acquisition. We aim to assess the safety and efficacy of the vaccine in South Africa, where the major circulating clade is subtype C.

METHODS

We did a phase 2b double-blind, randomised test-of-concept study in sexually active HIV-<em>1</em> seronegative participants at five sites in South Africa. Randomisation was by a computer-generated random number sequence. The vaccine and placebo were given by intramuscular injection on a 0, <em>1</em>, 6 month schedule. Our coprimary endpoints were a vaccine-induced reduction in HIV-<em>1</em> acquisition and viral-load setpoint. These endpoints were assessed independently in the modified intention-to-treat (MITT) cohort with two-tailed significance tests stratified by sex. We assessed immunogenicity by interferon-γ ELISPOT in peripheral-blood mononuclear cells. After the lack of efficacy of the MRKAd5 HIV-<em>1</em> vaccine in the Step study, enrolment and vaccination in our study was halted, treatment allocations were unmasked, and follow-up continued. This study is registered with the South Africa National Health Research Database, number DOH-27-0207-<em>1</em>539, and ClinicalTrials.gov, number NCT004<em>1</em>3725.

RESULTS

80<em>1</em> of a scheduled 3000 participants, of whom 360 (45%) were women, were randomly assigned to receive either vaccine or placebo. 445 participants (56%) had adenovirus serotype 5 (Ad5) titres greater than 200, and <em>1</em>29 men (29%) were circumcised. 34 MITT participants in the vaccine group were diagnosed with HIV-<em>1</em> (incidence rate 4·54 per <em>1</em>00 person-years) and 28 in the placebo group (3·70 per <em>1</em>00 person-years). There was no evidence of vaccine efficacy; the hazard ratio adjusted for sex was <em>1</em>·25 (95% CI 0·76-2·05). Vaccine efficacy did not differ by Ad5 titre, sex, age, herpes simplex virus type 2 status, or circumcision. The geometric mean viral-load setpoint was 20,483 copies per mL (n=33) in the vaccine group and 34,032 copies per mL (n=28) in the placebo group (p=0·39). The vaccine elicited interferon-γ-secreting T cells that recognised both clade B (89%) and C (77%) antigens.

CONCLUSIONS

The MRKAd5 HIV-<em>1</em> vaccine did not prevent HIV-<em>1</em> infection or lower viral-load setpoint; however, stopping our trial early probably compromised our ability to draw conclusions. The high incidence rates noted in South Africa highlight the crucial need for intensified efforts to develop an efficacious vaccine.

BACKGROUND

The US National Institute of Allergy and Infectious Disease and Merck and Co Inc.

Eight normal human lungs obtained from patients dying from causes not related to the lung were subjected to morphometric analysis to determine the number of cells in the alveolar region and their mean volume and surface characteristics. The age range was <em>1</em>9 to 40 yr, average body weight was 74 kg, and the average fixed lung volume was 4,300 <em>ml</em>. The overall mean nuclear diameters of the nuclei of 5 major cell types in the lung parenchyma were found to have little variation, with means ranging from 7.54 to 8.77 micrometers. Alveolar type I epithelial cells were found to comprise 8% of the cells and to be one of the largest cells, having a mean volume of <em>1</em>,764 micrometers 3 and covering an average of 5,098 micrometers 2 of alveolar surface. Seven percent of the alveolar surface was covered by alveolar type II cells, which make up <em>1</em>6% of the total alveolar cells and have a mean volume that is half that of the type I pneumocyte. Capillary endothelial cells make up 30% of the lung cells and were significantly smaller in both size and average surface area than the alveolar type I cells. Cells in the interstitial space comprised 37% of the total cells. The number of alveolar macrophages showed great variability, ranging from <em>1</em>9% of alveolar cells in <em>1</em> person to 3 to 5% in the nonsmoking females. The alveolar cell population characteristics found in resected lobes from 2 nonsmoking females were found to be similar to 2 nonsmoking females studied after autopsy. An interspecies comparison of characteristics of cells from the alveolar regions of normal lungs from humans, baboons, and rats showed that proportions of cells in the alveolar region and their average thickness, size, and surface areas were relatively constant.

Understanding the mechanisms by which adult stem cells produce growth factors may represent an important way to optimize their beneficial paracrine and autocrine effects. Components of the wound milieu may stimulate growth factor production to promote stem cell-mediated repair. We hypothesized that tumor necrosis factor-alpha (TNF-alpha), endotoxin (LPS), or hypoxia may activate human mesenchymal stem cells (MSCs) to increase release of vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), insulin-like growth factor <em>1</em> (IGF-<em>1</em>), or hepatocyte growth factor (HGF) and that nuclear factor-kappa B (NF kappa B), c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) mediates growth factor production from human MSCs. To study this, human MSCs were harvested, passaged, divided into four groups (<em>1</em>00,000 cells, triplicates) and treated as follows: <em>1</em>) with vehicle; 2) with stimulant alone [24 h LPS (200 ng/<em>ml</em>), 24 h TNF-alpha (50 ng/<em>ml</em>), or 24 h hypoxia (<em>1</em>% O2)]; 3) with inhibitor alone [NF kappa B (PDTC, <em>1</em> mM), JNK (TI-JIP, <em>1</em>0 microM), or ERK (ERK Inhibitor II, 25 microM)]; and 4) with stimulant and the various inhibitors. After 24 h incubation, MSC activation was determined by measuring supernatants for VEGF, FGF2, IGF-<em>1</em>, or HGF (ELISA). TNF-alpha, LPS, and hypoxia significantly increased human MSC VEGF, FGF2, HGF, and IGF-<em>1</em> production versus controls. Stem cells exposed to injury demonstrated increased activation of NF kappa B, ERK, and JNK. VEGF, FGF2, and HGF expression was significantly reduced by NF kappa B inhibition (50% decrease) but not ERK or JNK inhibition. Moreover, ERK, JNK, and NF kappa B inhibitor alone did not activate MSC VEGF expression over controls. Various stressors activate human MSCs to increase VEGF, FGF2, HGF, and IGF-<em>1</em> expression, which depends on an NFkB mechanism.

BACKGROUND

Over 150,000 Malawians have started antiretroviral therapy (ART), in which first-line therapy is stavudine/lamivudine/nevirapine. We evaluated drug resistance patterns among patients failing first-line ART on the basis of clinical or immunological criteria in Lilongwe and Blantyre, Malawi.

METHODS

Patients meeting the definition of ART failure (new or progressive stage 4 condition, CD4 cell count decline more than 30%, CD4 cell count less than that before treatment) from January 2006 to July 2007 were evaluated. Among those with HIV RNA of more than 1000 copies/ml, genotyping was performed. For complex genotype patterns, phenotyping was performed.

RESULTS

Ninety-six confirmed ART failure patients were identified. Median (interquartile range) CD4 cell count, log10 HIV-1 RNA, and duration on ART were 68 cells/microl (23-174), 4.72 copies/ml (4.26-5.16), and 36.5 months (26.6-49.8), respectively. Ninety-three percent of samples had nonnucleoside reverse transcriptase inhibitor mutations, and 81% had the M184V mutation. The most frequent pattern included M184V and nonnucleoside reverse transcriptase inhibitor mutations along with at least one thymidine analog mutation (56%). Twenty-three percent of patients acquired the K70E or K65R mutations associated with tenofovir resistance; 17% of the patients had pan-nucleoside resistance that corresponded to K65R or K70E and additional resistance mutations, most commonly the 151 complex. Emergence of the K65R and K70E mutations was associated with CD4 cell count of less than 100 cells/microl (odds ratio 6.1) and inversely with the use of zidovudine (odds ratio 0.18). Phenotypic susceptibility data indicated that the nucleoside reverse transcriptase inhibitor backbone with the highest activity for subsequent therapy was zidovudine/lamivudine/tenofovir, followed by lamivudine/tenofovir, and then abacavir/didanosine.

CONCLUSIONS

When clinical and CD4 cell count criteria are used to monitor first-line ART failure, extensive nucleoside reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor resistance emerges, with most patients having resistance profiles that markedly compromise the activity of second-line ART.

Listeria monocytogenes is an intracellular parasite that can readily infect the macrophage-like cell line J774 and the kidney epithelial cell PtK2. After being ingested, the organism escapes from the phagolysosome into the host-cell cytoplasm. N-(7-Nitrobenz-2-oxa-<em>1</em>,3-diazol-4-yl)-phallacidin, a specific stain for actin filaments (F-actin), demonstrates that within <em>1</em> hr of initiation of infection, the bacteria become surrounded by host-cell cytoplasmic actin filaments. By 3 hr, long projections of F-actin begin to form at one end of the bacteria. These actin structures colocalize with the actin-bundling protein alpha-actinin as well as with tropomyosin. Microinjection of fluorescently labeled alpha-actinin in living cells demonstrates that the formation of these F-actin projections is associated with bacterial movement, actin filaments rapidly assembling behind the bacteria as they migrate through the cytoplasm. These F-actin tails attain lengths up to 40 microns. The movement of the bacteria through the cytoplasm is rapid, 0.<em>1</em>2-<em>1</em>.46 microns/sec. Within 2 min of cytochalasin D (0.5 micrograms/<em>ml</em>) treatment, all bacterial intracellular movement stops, and additional bacteria-associated actin assembly is blocked. A nonmotile Listeria mutant induces comparable actin assembly and moves at speeds similar to the wild type, indicating that the forces required for intracellular bacterial movement are generated by the host cell. L. monocytogenes can dramatically stimulate host-cell actin assembly in a directional manner, which serves to rapidly propel the bacteria through the cytoplasm, allowing the organisms to move to peripheral membranes and spread to uninfected cells.