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Polymer-ceramic nanocomposites have been thoroughly investigated previously for high energy storage devices. However, the increase in performance of these nanocomposites has proven to be significantly lower than predicted values. Through surface functionalization of high dielectric constant nanoparticles (NP), the flaws that reduce composite performance can be eliminated to form high energy density composite materials. Functionalization methods utilize high throughput printing and curing techniques (i.e., inkjet printing and xenon flash lamp curing) that are crucial for rapid adoption into industrial production. (Ba,Ca) (Zr,Ti)O3 NPs (50 nm) are synthesized through the solvothermal method and functionalized with alkene terminated methoxysilanes. A thiol-ene monomer ink system, PTD3 [pentaerythritol tetrakis (3-mercaptopropionate) (PEMP, P), 1,3-Diisopropenylbenzene (DPB, D), 2,4,6-Triallyloxy-1,3,5-triazine (TOTZ, T)], is used as a high breakdown polymer matrix. Neat polymer, alkene terminated NP-polymer composites, and hydrophilic, TBAOH functionalized NP-polymer composites were spin coated onto both copper laminated glass slides and printed onto copper substrates in 1 cm(2) patterns for testing. Alkene functionalized NPs increased the breakdown strength by ∼38% compared to the nonfunctionalized NPs. Functionalized NPs increased both the breakdown strength and dielectric constant compared to the neat polymer, increasing the energy density nearly 3-fold from 13.3 to 36.1 J·cm(-3).

Artificial RNA switches are an emerging class of genetic controllers suitable for synthetic biology applications. Aptazymes are fusions composed of an aptamer domain and a self-cleaving ribozyme. The utilization of aptazymes for conditional gene expression displays several advantages over employing conventional transcription factor-based techniques as aptazymes require minimal genomic space, fulfill their function without the need of protein cofactors, and most importantly are reprogrammable with respect to ligand selectivity and the RNA function to be regulated. Technologies that enable the generation of aptazymes to defined input ligands are of interest for the construction of biocomputing devices and biosensing applications. In this chapter we present a method that facilitates the in vivo screening of randomized pools of aptazymes in mammalian cells.

Synthetic RNA-based switches are a growing class of genetic controllers applied in synthetic biology to engineer cellular functions. In this chapter, we detail a protocol for the selection of posttranscriptional controllers of gene expression in yeast using the Schistosoma mansoni hammerhead ribozyme as a central catalytic unit. Incorporation of a small molecule-sensing aptamer domain into the ribozyme renders its activity ligand-dependent. Aptazymes display numerous advantages over conventional protein-based transcriptional controllers, namely, the use of little genomic space for encryption, their modular architecture allowing for easy reprogramming to new inputs, the physical linkage to the message to be controlled, and the ability to function without protein cofactors. Herein, we describe the method to select ribozyme-based switches of gene expression in Saccharomyces cerevisiae that we successfully implemented to engineer neomycin- and theophylline-responsive switches. We also highlight how to adapt the protocol to screen for switches responsive to other ligands. Reprogramming of the sensor unit and incorporation into any RNA of interest enables the fulfillment of a variety of regulatory functions. However, proper functioning of the aptazyme is largely dependent on optimal connection between the aptamer and the catalytic core. We obtained functional switches from a pool of variants carrying randomized connection sequences by an in vivo selection in MaV203 yeast cells that allows screening of a large sequence space of up to 1×10(9) variants. The protocol given explains how to construct aptazyme libraries, carry out the in vivo selection and characterize novel ON- and OFF-switches., (© 2015 Elsevier Inc. All rights reserved.)

RNAs are ideal for the design of gene switches that can monitor and program cellular behavior because of their high modularity and predictable structure-function relationship. We have assembled an expression platform with an embedded modular ribozyme scaffold that correlates self-cleavage activity of designer ribozymes with transgene translation in bacteria and mammalian cells. A design approach devised to screen ribozyme libraries in bacteria and validate variants with functional tertiary stem-loop structures in mammalian cells resulted in a designer ribozyme with a protein-binding nutR-boxB stem II and a selected matching stem I. In a mammalian expression context, this designer ribozyme exhibited dose-dependent translation control by the N-peptide, had rapid induction kinetics and could be combined with classic small molecule-responsive transcription control modalities to construct complex, programmable genetic circuits.

Objective: To estimate the prevalence of ventilator-associated pneumonia caused by Pseudomonas aeruginosa in patients at risk for ventilator-associated pneumonia and to describe risk factors for P. aeruginosa ventilator-associated pneumonia., Design: Prospective, observational study., Setting: ICUs at 56 sites in 11 countries across four regions: the United States (n = 502 patients), Europe (n = 495), Latin America (n = 500), and Asia Pacific (n = 376)., Patients: Adults intubated and mechanically ventilated for 48 hours to 7 days, inclusive., Interventions: None (local standard of care)., Measurements and Main Results: Ventilator-associated pneumonia prevalence as defined by local investigators were 15.6% (293/1,873) globally, 13.5% in the United States, 19.4% in Europe, 13.8% in Latin America, and 16.0% in Asia Pacific (p = 0.04). Corresponding P. aeruginosa ventilator-associated pneumonia prevalences were 4.1%, 3.4%, 4.8%, 4.6%, and 3.2% (p = 0.49). Of 50 patients with P. aeruginosa ventilator-associated pneumonia who underwent surveillance testing, 19 (38%) had prior P. aeruginosa colonization and 31 (62%) did not (odds ratio, 7.99; 95% CI, 4.31-14.71). Of predefined risk factors for multidrug resistance (hereafter, risk factors), the most frequent in all patients were antimicrobial therapy within 90 days (51.9% of enrolled patients) and current hospitalization of more than or equal to 5 days (45.3%). None of these risk factors were significantly associated with P. aeruginosa ventilator-associated pneumonia by multivariate logistic regression. Risk factors associated with prior P. aeruginosa colonization were antimicrobial therapy within 90 days (odds ratio, 0.46; 95% CI, 0.29-0.73) and high proportion of antibiotic resistance in the community or hospital unit (odds ratio, 1.79; 95% CI, 1.14-2.82)., Conclusions: Our findings suggest that ventilator-associated pneumonia remains a common ICU infection and that P. aeruginosa is one of the most common causative pathogens. The odds of developing P. aeruginosa ventilator-associated pneumonia were eight times higher in patients with prior P. aeruginosa colonization than in uncolonized patients, which in turn was associated with local resistance.

A better understanding of changes in HIV-1 population genetics with combination antiretroviral therapy (cART) is critical for designing eradication strategies. We therefore analyzed HIV-1 genetic variation and divergence in patients' plasma before cART, during suppression on cART, and after viral rebound. Single-genome sequences of plasma HIV-1 RNA were obtained from HIV-1 infected patients prior to cART (N = 14), during suppression on cART (N = 14) and/or after viral rebound following interruption of cART (N = 5). Intra-patient population diversity was measured by average pairwise difference (APD). Population structure was assessed by phylogenetic analyses and a test for panmixia. Measurements of intra-population diversity revealed no significant loss of overall genetic variation in patients treated for up to 15 years with cART. A test for panmixia, however, showed significant changes in population structure in 2/10 patients after short-term cART (<1 year) and in 7/10 patients after long-term cART (1-15 years). The changes consisted of diverse sets of viral variants prior to cART shifting to populations containing one or more genetically uniform subpopulations during cART. Despite these significant changes in population structure, rebound virus after long-term cART had little divergence from pretherapy virus, implicating long-lived cells infected before cART as the source for rebound virus. The appearance of genetically uniform virus populations and the lack of divergence after prolonged cART and cART interruption provide strong evidence that HIV-1 persists in long-lived cells infected before cART was initiated, that some of these infected cells may be capable of proliferation, and that on-going cycles of viral replication are not evident.

In many synthetic biology applications, modular and easily accessible tools for controlling gene expression are required. In addition, in vivo biosensors and diagnostic devices will become more important in the future to allow for noninvasive determination of protein, ion, or small molecule metabolite levels. In recent years synthetic RNA-based switches have been developed to act as signal transducers to convert a binding event of a small molecule (input) into a detectable output. Their modular design allows the development of a variety of molecular switches to be used in biochemical assays or inside living cells. RNA switches developed by our group are based on the Schistosoma mansoni hammerhead ribozyme, a self-cleaving RNA sequence that can be inserted into any RNA of interest. Connection to an aptamer sensing a small molecule renders the cleavage reaction ligand-dependent. In the past we have successfully designed and applied such hammerhead aptazymes for the allosteric control of both bacterial and eukaryotic gene expression by affecting transcription elongation, translation initiation, or mRNA stability. In order to yield functional switches optimization of the connecting sequence between the aptamer and the HHR needs to be carried out. We have therefore developed an in vivo screening protocol detailed in this chapter that allows the identification of functional aptazymes in bacteria.

HIV infection is characterized by rapid and error-prone viral replication resulting in genetically diverse virus populations. The rate of accumulation of diversity and the mechanisms involved are under intense study to provide useful information to understand immune evasion and the development of drug resistance. To characterize the development of viral diversity after infection, we carried out an in-depth analysis of single genome sequences of HIV pro-pol to assess diversity and divergence and to estimate replicating population sizes in a group of treatment-naive HIV-infected individuals sampled at single (n = 22) or multiple, longitudinal (n = 11) time points. Analysis of single genome sequences revealed nonlinear accumulation of sequence diversity during the course of infection. Diversity accumulated in recently infected individuals at rates 30-fold higher than in patients with chronic infection. Accumulation of synonymous changes accounted for most of the diversity during chronic infection. Accumulation of diversity resulted in population shifts, but the rates of change were low relative to estimated replication cycle times, consistent with relatively large population sizes. Analysis of changes in allele frequencies revealed effective population sizes that are substantially higher than previous estimates of approximately 1,000 infectious particles/infected individual. Taken together, these observations indicate that HIV populations are large, diverse, and slow to change in chronic infection and that the emergence of new mutations, including drug resistance mutations, is governed by both selection forces and drift.

Synthetic biology approaches often combine natural building blocks to generate new cellular activities. Here, we make use of two RNA elements to design a regulatory device with novel functionality. The system is based on a hammerhead ribozyme (HHR) that cleaves itself to generate a liberated ribosome-binding site and, thus, permits expression of a downstream gene. We connected a temperature-responsive RNA hairpin to the HHR and, thus, generated a temperature-controlled ribozyme that we call thermozyme. Specifically, a Salmonella RNA thermometer (RNAT) known to modulate small heat shock gene expression by temperature-controlled base-pairing and melting was fused to the ribozyme. Following an in vivo screening approach, we isolated two functional thermozymes. In vivo expression studies and in vitro structure probing experiments support a mechanism in which rising temperatures melt the thermometer structure impairing the self-cleavage reaction of the ribozyme. Since RNA cleavage is necessary to liberate the RBS, these engineered thermozymes shut off gene expression in response to a temperature increase and, thus, act in a reverse manner as the natural RNAT. Our results clearly emphasize the highly modular nature and biotechnological potential of ribozyme-based RNA thermometers.

Background/aims: Microalbuminuria is a marker for early kidney disease and cardiovascular risk. The purposes of this study were to determine the prevalence of microalbuminuria in an HIV-infected clinic population, to test the predictive value of a single urine albumin/creatinine ratio (ACR) to identify persistent microalbuminuria and to examine covariates of microalbuminuria., Methods: We conducted a prospective cohort study of HIV-infected subjects (n = 182) without proteinuria (urine protein/creatinine ratio ≥0.5 g/g), elevated serum creatinine, diabetes, or chronic inflammatory conditions. Subjects completed three research visits within 9 months. Microalbuminuria was defined as the geometric mean ACR of 25-355 mg/g for females and 17-250 mg/g for males., Results: The prevalence of microalbuminuria was 14%. The negative predictive value of a single urine ACR determination was 98%, whereas the positive predictive value was only 74%. Microalbuminuria was similar among Black (15%) and non-Black (14%) subjects (p = 0.8). Subjects with microalbuminuria were more likely to have hypertension (p = 0.02) and metabolic syndrome (p = 0.03). While duration of HIV infection and the level of HIV viremia were similar between groups, those with microalbuminuria were more likely to have a CD4 count <200 cells/μl (p = 0.0003). In a multivariate logistic regression analysis, the only significant independent predictors of microalbuminuria were low CD4 count (p = 0.018) and current ritonavir exposure (p = 0.04)., Conclusion: The prevalence of microalbuminuria in an HIV-infected clinic population was similar to earlier reports, and was associated with hypertension and impaired immune function. A single normal ACR determination effectively excludes microalbuminuria, whereas an elevated ACR requires confirmation.

Background: The 2010 Dietary Guidelines emphasized that dietary sodium should be limited to 2300 mg/d, with a lower limit of 1500 mg/d for adults aged >50 y, non-Hispanic blacks, and those with diabetes, hypertension, or chronic kidney disease. The potassium goal remained at 4700 mg/d., Objective: The objective was to identify subpopulations for whom the 1500- or 2300-mg Na/d goals applied and to examine the joint sodium and potassium intakes for these persons., Design: The analyses were based on NHANES 2003-2008 data for 12,038 adult men and women aged ≥20 y. Persons aged >50 y, non-Hispanic blacks, and persons with hypertension, diabetes, and chronic kidney disease were identified. Mean sodium, potassium, and energy intakes were obtained from 2 nonconsecutive 24-h dietary recalls. Historical analyses of the sodium-potassium ratios in the American diet were based on NHANES 1971-2006., Results: Among persons recommended to consume <2300 mg Na/d, <0.12% jointly met the sodium and potassium guidelines. In the 1500-mg/d group, the guidelines were jointly met by <0.015%. Based on Dietary Guidelines, the corresponding dietary sodium-potassium ratio was either 0.49 (2300/4700) or 0.32 (1500/4700). Historical analyses of NHANES data by age-sex groups from 1971 to 2006 showed that sodium-potassium ratios were never <0.83., Conclusions: Joint dietary guidelines for sodium and potassium, intended for most American adults, are currently being met by ≤0.015% of the population. Extraordinary efforts will be needed to meet the recommendations of the Dietary Guidelines.

Background: Elevated serum interleukin 7 (IL-7) levels are observed in lymphopenic conditions, including idiopathic CD4 lymphopenia (ICL), which is characterized by CD4 lymphopenia in the absence of human immunodeficiency virus infection or other known immunodeficiency., Methods: To test whether defective IL-7 signaling could be an etiologic or contributing factor in ICL, peripheral blood mononuclear cells from patients with ICL (median CD4 T-cell count, 160 cells/μL) and healthy controls (median CD4 T-cell count, 582 cells/μL) were evaluated for expression of IL-7Rα chain (CD127) and intracellular phosphorylated STAT-5 (a marker of γc cytokine signaling) after cytokine stimulation. Gene expression was analyzed by real-time polymerase chain reaction following IL-7 stimulation., Results: The percentage of CD4+CD127+ T cells was lower in patients with ICL, compared with controls (P < .001). Lower levels of STAT-5 phosphorylation after IL-7 stimulation were observed in both CD4 and CD8 T cells from patients with ICL, compared with controls (P < .001 and P = .017, respectively), that inversely correlated in CD4 T cells with serum IL-7 levels (r = -0.734, P = .013). Destabilization of p27(kip1), a critical step for IL-7-induced T-cell cycling, was decreased in patients with ICL, compared with controls (P = .004), after IL-7 stimulation., Conclusions: These data suggest that diminished responsiveness to IL-7 in CD4 and CD8 T cells during ICL may be contributing to the dysregulation of T-cell homeostasis.

Background: Neisseria meningitidis is a leading cause of meningitis and septicemia globally. Recent shifts in serogroup dominance in some settings highlight the desirability of polysaccharide-conjugate vaccines with broader meningococcal coverage than serogroup C vaccines in widespread use., Methods: We assessed the safety and immunogenicity of a single dose of meningococcal quadrivalent (A, C, W-135, Y) tetanus conjugate vaccine (TetraMen-T), administered at 1 year of age. A total of 378 children were randomized to 1 of 6 groups--5 received alternative formulations of TetraMen-T, the sixth licensed adjuvanted serogroup C conjugate vaccine (Neisvac-C). Solicited adverse event reports were collected from day 0 to 7 after vaccination and unsolicited and serious adverse event reports throughout study participation. Immunogenicity was assessed by serum bactericidal assays containing either a human (hSBA) or baby rabbit (rSBA) complement source before and 1 month after immunization., Results: All vaccine formulations were safe and well tolerated. Using the various measures of immunogenicity, no consistent relationships were observed between the dose of either polysaccharide or carrier and serogroup-specific response for any one antigen. The highest-dose vaccine provided optimal coverage for all 4 serogroups, with the percentage of recipients achieving hSBA titers ≥ 8 against each as follows: A, 92%; C, 96%; W-135, 71%; Y, 82% (corresponding proportions with rSBAs titers >8 all exceeded 90%). The investigational vaccines were less immunogenic against the serogroup C capsular polysaccharide than the licensed comparator., Conclusions: Studies are ongoing that will help to identify optimal scheduling of quadrivalent meningococcal conjugate vaccines, to facilitate their inclusion into national immunization programs seeking extended serogroup coverage against meningococci.

Phototrophic epilithic biofilms harbour a distinct assemblage of heterotrophic bacteria, cyanobacteria and photoautotrophic algae. Secretion of extracellular polymeric substances (EPS) by these organisms and the physicochemical properties of the EPS are important factors for the development of the biofilms. We have isolated representative diatom and bacteria strains from epilithic biofilms of Lake Constance. By pairwise co-cultivating these strains we found that diatom growth and EPS secretion by diatoms may depend on the presence of individual bacteria. Similar results were obtained after addition of spent bacterial medium to diatom cultures, suggesting that soluble substances from bacteria have an impact on diatom physiology. While searching for putative bacterial signal substances, we found that concentrations of various dissolved free amino acids (DFAA) within the diatom cultures changed drastically during co-cultivation with bacteria. Further, the secretion of extracellular carbohydrates and proteins can be influenced by bacteria or their extracellular substances. We have performed mass spectrometric peptide mapping to identify proteins which are secreted when co-cultivating the diatom Phaeodactylum tricornutum Bohlin and Escherichia coli. The identified proteins are possibly involved in signalling, extracellular carbohydrate modification and uptake, protein and amino acid modification, and cell/cell aggregation of diatom and bacteria strains. Our data indicate that diatom-bacteria biofilms might be regulated by a complex network of chemical factors involving EPS, amino acid monomers and other substances. Thus interactions with bacteria can be considered as one of the main factors driving biofilm formation by benthic diatoms., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Background: 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins) exhibit antiviral activity against human immunodeficiency virus type 1 (HIV-1) in vitro and may modulate the immune response to HIV infection. Studies evaluating the antiviral activity of statins have yielded conflicting results., Methods: We conducted a randomized, double-blind, placebo-controlled crossover trial to investigate the effect of atorvastatin on HIV-1 RNA (primary objective) and cellular markers of immune activation (secondary objective). HIV-infected individuals not receiving antiretroviral therapy were randomized to receive either 8 weeks of atorvastatin (80 mg) or placebo daily. After a 4-6 week washout phase, participants switched treatment assignments. The study had 80% power to detect a 0.3 log(10) decrease in HIV-1 RNA level. Expression of CD38 and HLA-DR on CD4(+) and CD8(+) T cells was used to measure immune activation., Results: Of 24 randomized participants, 22 completed the study. Although HIV-1 RNA level was unaffected by the intervention (-0.13 log(10) copies/mL; P = .85), atorvastatin use resulted in reductions in circulating proportions of CD4(+) HLA-DR(+) (-2.5%; P = .02), CD8(+) HLA-DR(+) (-5%; P = .006), and CD8(+) HLA-DR(+) CD38(+) T cells (-3%; P = .03). Reductions in immune activation did not correlate with declines in serum levels of low-density lipoprotein cholesterol., Conclusions: Short-term use of atorvastatin was associated with modest but statistically significant reductions in the proportion of activated T lymphocytes.

Immune activation plays an important role in the pathogenesis of HIV disease. Although the causes are not fully understood, the forces that lead to immune dysfunction differ for CD4 and CD8 T cells. In this study, we report that the molecular pathways that drive immune activation during chronic HIV infection are influenced by differences in the homeostatic regulation of the CD4 and CD8 T cell pools. Proliferation of CD4 T cells is controlled more tightly by CD4 T cell numbers than is CD8 T cell proliferation. This difference reflects the importance of maintaining a polyclonal CD4 T cell pool in host surveillance. Both pools of T cells were found to be driven by viral load and its associated state of inflammation. In the setting of HIV-induced lymphopenia, naive CD4 T cells were recruited mainly into the proliferating pool in response to CD4 T cell depletion, whereas naive CD8 T cell proliferation was driven mainly by levels of HIV RNA. RNA analysis revealed increased expression of genes associated with type I IFN and common γ chain cytokine signaling in CD4 T cell subsets and only type I IFN-associated genes in CD8 T cell subsets. In vitro studies demonstrated enhanced STAT1 phosphorylation in response to IFN-α and increased expression of the IFNAR1 transcripts in naive and memory CD4 T cells compared with that observed in CD8 T cells. CD4 T cell subsets also showed enhanced STAT1 phosphorylation in response to exogenous IL-7.

Background: The pathogenesis of immunodeficiency due to human immunodeficiency virus (HIV)-1 is incompletely understood, but immune activation is believed to play a central role. Immunomodulatory agents that decrease immune activation may be useful in the treatment of HIV-1 infection., Methodology: A randomized, double blind, placebo-controlled pilot study of leflunomide for 28 days was performed in participants with HIV-1 infection who were not receiving antiretroviral therapy. Participants randomized to leflunomide were subsequently treated with cholestyramine until leflunomide levels were below detection limit., Findings: Treatment with leflunomide was well tolerated with mostly low-grade adverse events. Leflunomide administration reduced cycling of CD4 T cells (by ex vivo bromodeoxyuridine uptake and Ki67 expression) and decreased expression of activation markers (HLA-DR/CD38 co-expression) on CD8 T cells in peripheral blood. In addition, decreased expression of HIV-1 co-receptors was observed in both CD4 and CD8 T cells in the leflunomide group. There were no significant changes in naïve and memory T cell subsets, apoptosis of T cells or markers of microbial translocation., Conclusions: Leflunomide was effective in reducing immune activation in the setting of chronic HIV-1 infection suggesting that targeting immune activation with immunomodulatory agents may be a feasible strategy., Trial Registration: ClinicalTrials.gov NCT00101374.

Biomarkers could be useful in evaluating immune reconstitution inflammatory syndrome (IRIS). A cohort of 45 HIV-1-infected, antiretroviral treatment (ART)-naive patients with baseline CD4 T cell counts

Objective: Cardiovascular disease (CVD) contributes significantly to HIV-related morbidity and mortality. Chronic immune activation and inflammation are thought to augment the progression of atherosclerotic disease. In this retrospective, case-control study of HIV-infected individuals, we investigated the association of traditional cardiac risk factors, HIV-related disease, and inflammation with CVD events., Methods: HIV-infected individuals who experienced an incident CVD event while enrolled in National Institutes of Health clinical protocols from 1995 to 2009 were matched 2: 1 to HIV-infected individuals without known CVD. Markers of inflammation and cell activation were measured in serum or plasma using ELISA-based assays and peripheral mononuclear cells by four-color flow cytometry., Results: Fifty-two patients experienced an incident CVD event. Events were related to smoking, dyslipidemia, hyperglycemia, and family history as well as elevated D-dimer, soluble vascular cell adhesion molecule-1, tissue inhibitor of metalloproteinase-1, and soluble tissue factor, but not high-sensitivity C-reactive protein. No significant differences in antiviral therapy, CD4 T-cell count, or CD38 and human leukocyte antigen-DR expression were identified between patients and controls. In multivariable analysis, smoking, family history, D-dimer, and glucose were independently related to CVD risk., Conclusion: In this cohort, CVD risk was related to traditional CVD risk factors and markers of thrombosis and endothelial damage, but not to high-sensitivity C-reactive protein or markers of T-cell activation such as CD38/human leukocyte antigen-DR coexpression. D-dimer may help identify HIV-infected patients at elevated CVD risk.

Objective: To determine the effects of interleukin (IL)-2 treatment on inflammatory and thrombotic biomarkers in chronically HIV-infected adults receiving antiretroviral therapy., Methods: Cryopreserved plasma was evaluated retrospectively for C-reactive protein (CRP) and D-dimer at baseline, end of an IL-2 cycle, and long-term follow up from two randomized, controlled trials: 57 IL-2-naive adults receiving either three to six cycles of IL-2 as well as antiretroviral therapy (nucleoside analogues) or antiretroviral therapy alone for 12 months, and 40 IL-2-experienced adults on highly active antiretroviral therapy who either interrupted or continued therapy for 6 months after a baseline IL-2 cycle. High-sensitivity CRP (hsCRP) was measured by immunonephelometry (detection limit 0.175 mg/l) and D-dimer by latex agglutination (detection limit 0.20 mg/l). Median within-group differences and pre and post-IL-2 changes between groups were assessed via nonparametric Wilcoxon signed-rank and Mann-Whitney U-tests. Spearman's rank test was used to assess correlations between changes in hsCRP, D-dimer, and HIV-RNA viral load., Results: Significant increases in hsCRP (study 1: 138.6 mg/l; study 2: 58.9 mg/l) and D-dimer (study 1: 3.1 mg/l; study 2: 0.4 mg/l, all P < 0.0001) occurred by the end of the initial IL-2 cycle, returning to baseline by the end of study. No correlations were seen between changes in hsCRP or D-dimer and HIV-RNA, CD4 T-cell count, or proliferation (Ki67 expression). No thrombotic or cardiovascular serious adverse events occurred during these study periods., Conclusion: IL-2 dosing caused transient increases in plasma hsCRP and D-dimer levels, regardless of HIV-RNA viral load, suggesting the possibility of increased risk for thrombotic events.

Objectives: The goal of the present study was to determine the association between childhood overweight and area-based socioeconomic indicators in California Assembly districts., Design: A cross-sectional ecologic study., Participants: California public school students., Main Exposure: Poverty and demographic data for California Assembly districts were based on the 2000 Census and obtained from the UCLA Center for Health Policy Research., Outcome Measures: Overall and race- and ethnicity-specific rates of childhood overweight for California Assembly districts (n=80) were based on the 2004 statewide Fitnessgram evaluation of California public school students., Results: Poverty was significantly associated with childhood overweight in California Assembly districts. At the Assembly district scale, childhood overweight was significantly associated with percent residents below poverty for the entire population (r=0.82), and with the race/ethnicity-specific overweight prevalence for African-American (r=0.43), Latino (r=0.61) and White (r=0.54) populations. There was also evidence that childhood overweight in California Assembly districts was spatially clustered. Linear regression models confirmed that percent of residents below poverty was an independent predictor of a higher prevalence of childhood overweight for the entire population. The results of race/ethnicity-specific models confirmed that the association between area poverty and childhood overweight was not explained by differences in the risk of overweight among specific race/ethnicity groups., Conclusions: Area-based measures of socioeconomic status can be used to identify problem areas and can be used for optimal targeting of public health prevention and intervention efforts.

Virus-specific CD8+ T cells probably mediate control over HIV replication in rare individuals, termed long-term nonprogressors (LTNPs) or elite controllers. Despite extensive investigation, the mechanisms responsible for this control remain incompletely understood. We observed that HIV-specific CD8+ T cells of LTNPs persisted at higher frequencies than those of treated progressors with equally low amounts of HIV. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This function required lytic granule loading of effectors and delivery of granzyme B to target cells. Defective cytotoxicity of progressor effectors could be restored after treatment with phorbol ester and calcium ionophore. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. They also demonstrate that lytic granule contents of memory cells are a critical determinant of cytotoxicity that must be induced for maximal per-cell killing capacity.

HIV infection is characterized by a brisk immune activation that plays an important role in the CD4 depletion and immune dysfunction of patients with AIDS. The mechanism underlying this activation is poorly understood. In the current study, we tested the hypothesis that this activation is the net product of two distinct pathways: the inflammatory response to HIV infection and the homeostatic response to CD4 T cell depletion. Using ex vivo BrdU incorporation of PBMCs from 284 patients with different stages of HIV infection, we found that CD4 proliferation was better predicted by the combination of CD4 depletion and HIV viral load (R(2) = 0.375, P < 0.001) than by either parameter alone (CD4 T cell counts, R(2) = 0.202, P < 0.001; HIV viremia, R(2) = 0.302, P < 0.001). Interestingly, CD8 T cell proliferation could be predicted by HIV RNA levels alone (R(2) = 0.334, P < 0.001) and this predictive value increased only slightly (R(2) = 0.346, P < 0.001) when CD4 T cell depletion was taken into account. Consistent with the hypothesis that CD4 T cell proliferation is driven by IL-7 as a homeostatic response to CD4 T cell depletion, levels of phosphorylated STAT-5 were found to be elevated in naive subsets of CD4 and CD8 T cells from patients with HIV infection and in the central memory subset of CD4 T cells. Taken together these data demonstrate that at least two different pathways lead to immune activation of T cells in patients with HIV infection and these pathways differentially influence CD4 and CD8 T cell subsets.

A clear understanding of the antiviral effects of CD8(+) T cells in the context of chronic human immunodeficiency virus (HIV) infection is critical for the development of prophylactic vaccines and therapeutics designed to support T-cell-mediated immunity. However, defining the potential correlates of effective CD8(+) T-cell immunity has proven difficult; notably, comprehensive analyses have demonstrated that the size and shape of the CD8(+) T-cell response are not necessarily indicative of efficacy determined by measures of plasma viral load. Here, we conducted a detailed quantitative and qualitative analysis of CD8(+) T-cell responses to autologous virus in a cohort of six HIV-infected individuals with a history of structured interruption of antiretroviral therapy (ART) (SIT). The magnitude and breadth of the HIV-specific response did not, by themselves, explain the changes observed in plasma virus levels after the cessation of ART. Furthermore, mutational escape from targeted epitopes could not account for the differential virological outcomes in this cohort. However, the functionality of HIV-specific CD8(+) T-cell populations upon antigen encounter, determined by the simultaneous and independent measurement of five CD8(+) T-cell functions (degranulation and gamma interferon, macrophage inflammatory protein 1beta, tumor necrosis factor alpha, and interleukin-2 levels) reflected the emergent level of plasma virus, with multiple functions being elicited in those individuals with lower levels of viremia after SIT. These data show that the quality of the HIV-specific CD8(+) T-cell response, rather than the quantity, is associated with the dynamics of viral replication in the absence of ART and suggest that the effects of SIT can be assessed by measuring the functional profile of HIV-specific CD8(+) T cells.

Current antiretroviral therapy is effective in suppressing but not eliminating HIV-1 infection. Understanding the source of viral persistence is essential for developing strategies to eradicate HIV-1 infection. We therefore investigated the level of plasma HIV-1 RNA in patients with viremia suppressed to less than 50-75 copies/ml on standard protease inhibitor- or non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy using a new, real-time PCR-based assay for HIV-1 RNA with a limit of detection of one copy of HIV-1 RNA. Single copy assay results revealed that >80% of patients on initial antiretroviral therapy for 60 wk had persistent viremia of one copy/ml or more with an overall median of 3.1 copies/ml. The level of viremia correlated with pretherapy plasma HIV-1 RNA but not with the specific treatment regimen. Longitudinal studies revealed no significant decline in the level of viremia between 60 and 110 wk of suppressive antiretroviral therapy. These data suggest that the persistent viremia on current antiretroviral therapy is derived, at least in part, from long-lived cells that are infected prior to initiation of therapy.

Purpose: To compare MRI findings and histological plasmacellular infiltration of the bone marrow in patients with multiple myeloma (MM)., Material and Methods: Twenty-four patients with different stages of MM underwent 1.5T MRI of the pelvic bone before iliac crest punch biopsy. Precontrast T1wSE and STIR and postcontrast (Gd-DTPA) T1wSE-fatsat were acquired using axial slices. Immediately after the biopsy, T1wSE was repeated to locate the biopsy canal. The corresponding region in the examination before punch biopsy was assessed for bone marrow involvement using a three-point score (0: negative, 1: suspect, 2: definite)., Results: Two patients were not included because the location of the biopsy canal was unclear. Of 7 patients without histological plasmacellular infiltration, MRI was false positive in one case (suspect). Of 15 patients with histological infiltration, MRI was positive in 10 cases (4 suspect, 6 definite). The T1wSE was positive in 9 cases, STIR in 8 cases, and postcontrast T1wSE-fatsat in 7 cases. In 10 of the 15 patients, the infiltration was histologically graded as low (5 - 20 % of bone marrow). In this group, MRI was only positive in 5 cases (3 suspect, 2 definite). Of five patients with the infiltration histologically graded as high (> 20 % of bone marrow), MRI was positive in all cases (1 suspect, 4 definite)., Conclusion: Only advanced bone marrow infiltration in MM can be reliably detected by MRI. None of the used sequences proved to be significantly superior or inferior.

The improved technical sophistication of laparoscopy offers an attractive highly accurate diagnostic modality at the bedside for critically ill patients who are confined to the ICU and intensive monitoring unit and who are in need of timely abdominal evaluation. If the surgeon understands the physiologic effects of peritoneal insufflation and considers them during the procedure, the patient tolerates the procedure well. It is important to limit insufflation pressures and laparoscopy time. The author also recommends using the open Hasson technique. Its primary use is diagnostic in penetrating and blunt trauma and in the obscure abdomen. It has been successfully employed therapeutically for acalculous cholecystitis, abscess drainage, and correction of placement of gastrostomy tubes and peritoneal dialysis catheters. It helps avoid risky transport trips and negative or nontherapeutic laparotomy with its known associated risks.

The technology to perform diagnostic and therapeutic procedures at the bedside continues to advance. Because of documented hazards and the expense of intrahospital transport, the bedside is becoming an appealing site for procedures that are more commonly performed in radiologic, bronchoscopic, other procedural suites, and the operating room.

Background: Anesthetic standard of care is to restrict oral intake for 8 hours before elective surgery. There is no research addressing appropriate preoperative discontinuation of jejunostomy tube (J-tube) feedings. We hypothesized that patients could be fed safely, via a J-tube, until the time of surgery., Methods: Patients admitted to a Level I Trauma Center, having J-tubes and undergoing a nonabdominal operation, were prospectively evaluated. Group I patients received J-tube feedings until transport to the operating room. Group II patients had tube feedings discontinued for at least 8 hours before surgery. Data were compared using the Student's t test and contingency table analysis., Results: There were 46 patients in group I and 36 in group II. There was no incidence of aspiration. Patient groups did not differ in age, mortality, length of stay, injury severity score, or ventilator days. Group I patients had tube feedings discontinued for fewer hours before and after surgery than group II patients (before surgery: 1.40 +/- 1.20 vs 11.61 +/- 5.01, respectively; p < .001; after surgery: 2.99 +/- 7.49 vs 7.11 +/- 9.03, respectively; p = .043); received more kilocalories/ grams of protein on the day of surgery (group I vs group II, 1676.15/89.57 +/- 1133.21/38.04 vs 791.14/57.58 +/-498.66/79.87, respectively; p = .001/p = .032) and more kilocalories/grams of protein on the first postoperative day (group I vs group II, 1580.74/92.57 +/- 600.53/37.96 vs 1152.47/63.53 +/- 733.96/39.40, respectively; p = .006/p = .001)., Conclusions: Patients receiving J-tubes who are undergoing nonabdominal operations may safely continue enteral nutrition at maximum protein and caloric intake until surgery.

A significant number of elderly drivers present without discernible external cause for their road crash. We hypothesize that syncope may contribute to their crash etiology and prospectively evaluated drivers older than 60 years with unexplained crash, presenting to our trauma center during a 1-year period. Prehospital data and prior medical history were obtained on all, followed by a standard syncope work-up. Of 79 drivers ages 60-98, 58 (73%) were at fault in the crash. In 33 (57%), the crash etiology could not be determined. Thirteen had prior syncope history. Workup was negative in two patients, and six died or were transferred before evaluation. Twenty-five of 33 (76%) had positive workups for syncope: cardiac causes in 10; neurologic in seven; psychiatric in five, endocrine in two; one indeterminant. Ten of 18 who survived had license revocation for medical reasons. Syncopal episodes are a frequent contributor to unexplained crashes among elderly drivers and should be suspected whenever external causes are not apparent.

The ability of elderly citizens to drive safely has been the subject of ongoing debate. To identify the type of elderly driver who becomes involved in an injury-producing road crash, we profiled all drivers over 39 years of age admitted to our Level I Trauma Center over a 1-year period. Data were prospectively collected and drivers age 40-59 years were compared with those over 60 years. Eighty-four drivers age > or = 60 and 130 drivers age 40-59 were studied. Of the 84 elderly drivers, 67 were deemed at fault. Twenty-four of those crashes were due to driving errors, 12 due to syncopes, and in 20 no crash cause was determined. Fifty-three of the 67 at fault drivers had significant underlying medical problems, compared to 9 of 17 deemed not at fault. Only four were legally intoxicated. Of the 130 drivers in the comparison group, only 19 had significant underlying medical problems; in three syncope was suspected; 18 were legally intoxicated. We conclude from our data that underlying medical disorders occur frequently in elderly drivers and may contribute to their incidence of road crashes.

Purpose: This article assessed the value of routine head computerized axial tomographic (CT) scans for diagnosis of unsuspected facial fractures and its clinical implications in the multiply injured patient who is intubated, unconscious, or sedated at the time of initial assessment and requires a head CT scan to assess for brain injury., Methods: At a level I trauma center from June 1, 1992 to June 1, 1993 all intubated blunt trauma patients who required routine CT scan evaluation at initial assessment were studied prospectively. Routine scanning started at the foramen magnum and included the maxilla. Patients who died within the first 24 hours were excluded., Results: The study population included 116 patients (85 male, 21 female) aged 12 to 85 years (mean, 28 years) with injury severity scores ranging from 1 to 50 (mean, 23). The mechanism of injury was: motor vehicle accidents (n = 74), motorcycling (n = 5), pedestrians accidents (n = 13), falls (n = 10), bicycling (n = 5), assaults (n = 8), and boating accident (n = 1). There were 19 suspected facial fractures; 18 required surgical repair. There were 27 unsuspected facial fractures; 13 required surgical care. Three suspected fractures were ruled out., Conclusion: Routine head CT scans to assess for brain injury in the multiply injured patient are also very useful in the diagnosis of unsuspected facial fractures, almost half of which will require surgical intervention.

Study Objective: To evaluate the investigation and prosecution of drunk drivers and identify reasons for system failure., Design: Prospective data collection on all drivers with an elevated blood alcohol level who were treated at a Level I trauma center between January 1 and June 30, 1991., Setting: Level I trauma center/university medical center serving a population of 1.8 million., Interventions: Prospective data collection, interviewing ambulance squad members, investigating police, and results of prosecution., Measurements and Main Results: Of 321 drivers, 78 had ethanol levels of more than 100 mg/dL; nine were between 50 and 100 mg/dL. All drivers had rapid transport to a trauma center for severe injury or high-risk injury mechanism. Police reports were accessible for 84 of 87 drivers; all drivers were believed to be at fault. The crashes resulted in five deaths and 74 other victims requiring hospitalization. Six of the intoxicated drivers died. Police requests for blood alcohol levels were made for 28 drivers, one of whom died. The remaining 59 drivers had no legal blood alcohol level drawn. Although the greater the distance from our center (more rural), the less likely were requests for ethanol levels, but there were many "no requests" from nearby cities. Of the 28 drivers on whom legal alcohol levels were drawn, eight escaped central registration with the highway safety commission, which records any and every traffic violation within New Jersey. Of the remaining 20, eight were not prosecuted (excluding one death), including one repeat offender. One committed a later "driving while intoxicated" offense and was then prosecuted. The remaining 11 were all convicted; five were repeat offenders., Conclusion: Reasonable cause is required for legal blood alcohol requests. Our data demonstrated that this is difficult when the driver is unavailable for questioning. However, once legal blood alcohol levels have been obtained, inadequate legal follow-up leads to nonprosecution. Once officially charged, conviction appears certain, but even this punishment and re-education fail to change behavior in many of these drivers.

The increased coagulability in multiple injured patients results in increased risk for deep venous thrombosis and pulmonary embolism. The authors discuss etiology, diagnosis, treatment, and prophylaxis of venous thromboembolism in trauma patients.

Study Objective: We evaluated the importance of microscopic and gross hematuria and the role of retrograde cystography and computed tomography (CT) in the diagnosis of blunt traumatic bladder rupture., Design: All cases at a Level I trauma center between January 1, 1986, and March 31, 1989, were reviewed retrospectively., Setting: Level I trauma center, university hospital., Type of Participants: All patients with acute blunt abdominal trauma admitted to this Level I trauma center., Interventions: The patients' charts were reviewed with emphasis on mode of diagnosis, treatment, and outcome., Measurements and Main Results: Twenty-one patients had bladder rupture. All 21 had hematuria with more than 50 RBCs/high-power field, 17 gross and four microscopic. Twenty patients underwent retrograde cystography, which accurately identified bladder rupture, and one was found at laparotomy for other injuries. Seven patients had CT of the abdomen and pelvis, which failed to demonstrate bladder rupture. There were no associated urethral injuries in any of the patients with bladder rupture., Conclusion: Significant (more than 50 RBCs/high-power field) hematuria is the principal indication for evaluation for blunt bladder injury, and retrograde cystography is the diagnostic procedure of choice. CT is neither sensitive nor specific enough as primary diagnostic modality.

Not every patient with a penetrating stab wound of the abdomen requires laparotomy. This report evaluates use of computerized tomographic (CT) scan in assessment of stable asymptomatic patients, with penetrating abdominal stab wounds, as an indicator of the necessity of abdominal exploration. In a prospective study, 50 patients with abdominal stab wounds were treated successfully with observation only, after admission abdominal CT scan interpretation was negative for pathology in 45 patients. In the remaining five it was of such minor nature that conservative management was justified (Series I). Twenty-eight stable asymptomatic patients with penetrating stab wounds of the abdomen comparable to the ones in Series I had CT scan on admission, and then underwent exploratory laparotomy independent of their CT scan findings (Series II). Of these 28 patients, 22 had correct CT scan findings verified by laparotomy, three were false positive for intra-abdominal injury resulting in negative explorations, and three patients had such nonspecific findings as fluid or air in the abdomen incompatible with precise organ injury identification. Our study shows that CT scan in patients with abdominal stab wounds identifies solid organ injury with great specificity and sensitivity, evaluates the retroperitoneum well, and detects peritoneal penetration by demonstrating intraperitoneal fluid or air. CT scan was unreliable in detection of bowel injury and does not demonstrate diaphragmatic injuries.

Bullet emboli are rare and their management when in the venous circulation is controversial. A 26-year-old female with a gunshot wound to the heart, followed by embolization of the bullet to the right hepatic vein, had successful percutaneous retrieval of the bullet via a catheter inserted through the right femoral vein.