Analgesic efficacy of ultrasound-guided regional anesthesia: a meta-analysis.

STUDY OBJECTIVE: To determine if the use of ultrasound guidance (vs non-ultrasound techniques) improves the success rate of nerve blocks. DESIGN: Meta-analysis of randomized controlled trials (RCTs) in the published literature. SETTING: University medical center. MEASUREMENTS: 16 RCTs of patients undergoing elective surgical procedures were studied. Patients underwent ultrasound-guided or non-ultrasound techniques (nerve stimulation, surface landmark) for peripheral nerve blocks. Success rates were measured. MAIN RESULTS: Ultrasound guidance (vs all non-ultrasound techniques) was associated with a significant increase in the success rate of nerve blocks [relative risk (RR) = 1.11 (95% confidence interval [CI]: 1.06 to 1.17, P < 0.0001]). When compared with nerve stimulator techniques only, ultrasound guidance was still associated with an increase in the success rate (RR = 1.11 [95% CI: 1.05 to 1.17, P = 0.0001]). For specific blocks, ultrasound guidance (vs all non-ultrasound) was associated with a significant increase in successful brachial plexus (all) nerve blocks (RR = 1.11 [95% CI: 1.05 to 1.20, P = 0.0001]), sciatic popliteal nerve block (RR = 1.22 [95% CI: 1.08 to 1.39, P = 0.002]) and brachial plexus axillary nerve block (RR = 1.13 [95% CI: 1.00 to 1.26, P = 0.05]) but not brachial plexus infraclavicular nerve block (RR = 1.25 [95% CI: 0.88 to 1.76, P = 0.22]). CONCLUSIONS: Ultrasound-guided peripheral nerve block is associated with an increased overall success rate when compared with nerve stimulation or other methods. Ultrasound-guided techniques also increase the success rate of some specific blocks.

Modulation of DNA vaccine-elicited CD8+ T-lymphocyte epitope immunodominance hierarchies.

Generating broad cellular immune responses against a diversity of viral epitopes is a major goal of current vaccine strategies for human immunodeficiency virus type 1 (HIV-1) and other pathogens. Virus-specific CD8(+) T-lymphocyte responses, however, are often highly focused on a very limited number of immunodominant epitopes. For an HIV-1 vaccine, the breadth of CD8(+) T-lymphocyte responses may prove to be critical as a result of the need to cover a wide diversity of viral isolates in the population and to limit viral escape from dominant epitope-specific T lymphocytes. Here we show that epitope modification strategies can alter CD8(+) T-lymphocyte epitope immunodominance hierarchies elicited by a DNA vaccine in mice. Mice immunized with a DNA vaccine expressing simian immunodeficiency virus Gag lacking the dominant D(b)-restricted AL11 epitope generated a marked and durable augmentation of responses specific for the subdominant D(b)-restricted KV9 epitope. Moreover, anatomic separation strategies and heterologous prime-boost regimens generated codominant responses against both epitopes. These data demonstrate that dominant epitopes can dramatically suppress the immunogenicity of subdominant epitopes in the context of gene-based vaccines and that epitope modification strategies can be utilized to enhance responses to subdominant epitopes.

Evaluation of CD62L expression as a marker for vaccine-elicited memory cytotoxic T lymphocytes.

The development of successful vaccination strategies for eliciting cytotoxic T lymphocytes (CTLs) will be facilitated by the definition of strategies for subdividing CTLs into functionally distinct subpopulations. We assessed whether surface expression of a number of cell-surface proteins could be used to define functionally distinct subpopulations of memory CTLs in mice immunized with a recombinant vaccinia virus expressing human immunodeficiency virus (HIV)-1 envelope (Env). We found changes in cell-surface expression of CD11a, CD44, CD45RB, CD49d, CD54 and CD62L on Env-specific CD8(+) T cells that appeared to differentiate them from other CD8(+) T cells within 1 week to 1 month following immunization. Further, we saw an up-regulation of CD62L surface expression on Env-specific CD8(+) memory T cells several months after immunization. However, CD62L expression did not correlate with differences in the abilities of CTLs to proliferate or produce interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) in vitro in response to Env peptide stimulation. Moreover, the expression of CD62L did not allow differentiation of CTLs into subpopulations with distinct expansion kinetics in vivo after adoptive transfer into naïve mice and subsequent boosting of these mice with a recombinant adenovirus expressing HIV-1 Env. Therefore, the definition of memory CD8(+) T-cell subpopulations on the basis of CD62L expression in mice does not allow the delineation of functionally distinct CTL subpopulations.

Immunogenicity of recombinant fiber-chimeric adenovirus serotype 35 vector-based vaccines in mice and rhesus monkeys.

Preexisting immunity to adenovirus serotype 5 (Ad5) has been shown to suppress the immunogenicity of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 (HIV-1) in both preclinical studies and clinical trials. A potential solution to this problem is to utilize rAd vectors derived from rare Ad serotypes, such as Ad35. However, rAd35 vectors have appeared less immunogenic than rAd5 vectors in preclinical studies to date. In this study, we explore the hypothesis that the differences in immunogenicity between rAd5 and rAd35 vectors may be due in part to differences between the fiber proteins of these viruses. We constructed capsid chimeric rAd35 vectors containing the Ad5 fiber knob (rAd35k5) and compared the immunogenicities of rAd5, rAd35k5, and rAd35 vectors expressing simian immunodeficiency virus Gag and HIV-1 Env in mice and rhesus monkeys. In vitro studies demonstrated that rAd35k5 vectors utilized the Ad5 receptor CAR rather than the Ad35 receptor CD46. In vivo studies showed that rAd35k5 vectors were more immunogenic than rAd35 vectors in both mice and rhesus monkeys. These data suggest that the Ad5 fiber knob contributes substantially to the immunogenicity of rAd vectors. Moreover, these studies demonstrate that capsid chimeric rAd vectors can be constructed to combine beneficial immunologic and serologic properties of different Ad serotypes.

Immunogenicity of heterologous prime-boost regimens involving recombinant adenovirus serotype 11 (Ad11) and Ad35 vaccine vectors in the presence of anti-ad5 immunity.

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations will likely limit the immunogenicity and clinical utility of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. A potential solution to this problem is to utilize rAd vaccine vectors derived from rare Ad serotypes such as Ad35 and Ad11. We have previously reported that rAd35 vectors were immunogenic in the presence of anti-Ad5 immunity, but the immunogenicity of heterologous rAd prime-boost regimens and the extent that cross-reactive anti-vector immunity may limit this approach have not been fully explored. Here we assess the immunogenicity of heterologous vaccine regimens involving rAd5, rAd35, and novel rAd11 vectors expressing simian immunodeficiency virus Gag in mice both with and without anti-Ad5 immunity. Heterologous rAd prime-boost regimens proved significantly more immunogenic than homologous regimens, as expected. Importantly, all regimens that included rAd5 were markedly suppressed by anti-Ad5 immunity. In contrast, rAd35-rAd11 and rAd11-rAd35 regimens elicited high-frequency immune responses both in the presence and in the absence of anti-Ad5 immunity, although we also detected clear cross-reactive Ad35/Ad11-specific humoral and cellular immune responses. Nevertheless, these data suggest the potential utility of heterologous rAd prime-boost vaccine regimens using vectors derived from rare human Ad serotypes.

A human T-cell leukemia virus type 1 regulatory element enhances the immunogenicity of human immunodeficiency virus type 1 DNA vaccines in mice and nonhuman primates.

Plasmid DNA vaccines elicit potent and protective immune responses in numerous small-animal models of infectious diseases. However, their immunogenicity in primates appears less potent. Here we investigate a novel approach that optimizes regulatory elements in the plasmid backbone to improve the immunogenicity of DNA vaccines. Among various regions analyzed, we found that the addition of a regulatory sequence from the R region of the long terminal repeat from human T-cell leukemia virus type 1 (HTLV-1) to the cytomegalovirus (CMV) enhancer/promoter increased transgene expression 5- to 10-fold and improved cellular immune responses to human immunodeficiency virus type 1 (HIV-1) antigens. In cynomolgus monkeys, DNA vaccines containing the CMV enhancer/promoter with the HTLV-1 R region (CMV/R) induced markedly higher cellular immune responses to HIV-1 Env from clades A, B, and C and to HIV-1 Gag-Pol-Nef compared with the parental DNA vaccines. These data demonstrate that optimization of specific regulatory elements can substantially improve the immunogenicity of DNA vaccines encoding multiple antigens in small animals and in nonhuman primates. This strategy could therefore be explored as a potential method to enhance DNA vaccine immunogenicity in humans.

Neutralizing antibodies to adenovirus serotype 5 vaccine vectors are directed primarily against the adenovirus hexon protein.

The utility of recombinant adenovirus serotype 5 (rAd5) vector-based vaccines for HIV-1 and other pathogens will likely be limited by the high prevalence of pre-existing Ad5-specific neutralizing Abs (NAbs) in human populations. However, the immunodominant targets of Ad5-specific NAbs in humans remain poorly characterized. In this study, we assess the titers and primary determinants of Ad5-specific NAbs in individuals from both the United States and the developing world. Importantly, median Ad5-specific NAb titers were >10-fold higher in sub-Saharan Africa compared with the United States. Moreover, hexon-specific NAb titers were 4- to 10-fold higher than fiber-specific NAb titers in these cohorts by virus neutralization assays using capsid chimeric viruses. We next performed adoptive transfer studies in mice to evaluate the functional capacity of hexon- and fiber-specific NAbs to suppress the immunogenicity of a prototype rAd5-Env vaccine. Hexon-specific NAbs were remarkably efficient at suppressing Env-specific immune responses elicited by the rAd5 vaccine. In contrast, fiber-specific NAbs exerted only minimal suppressive effects on rAd5 vaccine immunogenicity. These data demonstrate that functionally significant Ad5-specific NAbs are directed primarily against the Ad5 hexon protein in both humans and mice. These studies suggest a potential strategy for engineering novel Ad5 vectors to evade dominant Ad5-specific NAbs.

Recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid DNA vaccines.

DCs are critical for priming adaptive immune responses to foreign antigens. However, the utility of harnessing these cells in vivo to optimize the immunogenicity of vaccines has not been fully explored. Here we investigate a novel vaccine approach that involves delivering synergistic signals that both recruit and expand DC populations at the site of antigen production. Intramuscular injection of an unadjuvanted HIV-1 envelope (env) DNA vaccine recruited few DCs to the injection site and elicited low-frequency, env-specific immune responses in mice. Coadministration of plasmids encoding the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) and the DC-specific growth factor fms-like tyrosine kinase 3 ligand with the DNA vaccine resulted in the recruitment, expansion, and activation of large numbers of DCs at the site of inoculation. Consistent with these findings, coadministration of these plasmid cytokines also markedly augmented DNA vaccine---elicited cellular and humoral immune responses and increased protective efficacy against challenge with recombinant vaccinia virus. These data suggest that the availability of mature DCs at the site of inoculation is a critical rate-limiting factor for DNA vaccine immunogenicity. Synergistic recruitment and expansion of DCs in vivo may prove a practical strategy for overcoming this limitation and potentiating immune responses to vaccines as well as other immunotherapeutic strategies.

Immunogenicity of recombinant adenovirus serotype 35 vaccine in the presence of pre-existing anti-Ad5 immunity.

The high prevalence of pre-existing immunity to adenovirus serotype 5 (Ad5) in human populations may substantially limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for HIV-1 and other pathogens. A potential solution to this problem is to use vaccine vectors derived from adenovirus (Ad) serotypes that are rare in humans, such as Ad35. However, cross-reactive immune responses between heterologous Ad serotypes have been described and could prove a major limitation of this strategy. In particular, the extent of immunologic cross-reactivity between Ad5 and Ad35 has not previously been determined. In this study we investigate the impact of pre-existing anti-Ad5 immunity on the immunogenicity of candidate rAd5 and rAd35 vaccines expressing SIV Gag in mice. Anti-Ad5 immunity at levels typically found in humans dramatically blunted the immunogenicity of rAd5-Gag. In contrast, even high levels of anti-Ad5 immunity did not substantially suppress Gag-specific cellular immune responses elicited by rAd35-Gag. Low levels of cross-reactive Ad5/Ad35-specific CD4(+) T lymphocyte responses were observed, but were insufficient to suppress vaccine immunogenicity. These data demonstrate the potential utility of Ad35 as a candidate vaccine vector that is minimally suppressed by anti-Ad5 immunity. Moreover, these studies suggest that using Ad vectors derived from immunologically distinct serotypes may be an effective and general strategy to overcome the suppressive effects of pre-existing anti-Ad immunity.

Recruitment of different subsets of antigen-presenting cells selectively modulates DNA vaccine-elicited CD4+ and CD8+ T lymphocyte responses.

The immunogenicity of plasmid DNA vaccines may be limited by the availability of professional antigen-presenting cells (APC) at the site of inoculation. Here we demonstrate that the types of APC recruited to the injection site can selectively modulate CD4(+) or CD8(+) T lymphocyte responses elicited by an HIV-1 Env DNA vaccine in mice. Coadministration of plasmid GM-CSF with the DNA vaccine resulted in the recruitment of macrophages to the site of inoculation and specifically augmented vaccine-elicited CD4(+) T lymphocyte responses. In contrast, coadministration of plasmid MIP-1 alpha with the DNA vaccine resulted in the recruitment of dendritic cells to the injection site and enhanced vaccine-elicited CD8(+) T lymphocyte responses. Interestingly, coadministration of both plasmid GM-CSF and plasmid MIP-1 alpha with the DNA vaccine recruited both macrophages and dendritic cells and led to a synergistic and sustained augmentation of CD4(+)and CD8(+) T lymphocyte responses. These data demonstrate the critical importance of locally recruited professional APC in determining the magnitude and nature of immune responses elicited by plasmid DNA vaccines. Moreover, these studies show that different subsets of professional APC can selectively modulate DNA vaccine-elicited T lymphocyte responses.

Neutralizing antibodies and CD8+ T lymphocytes both contribute to immunity to adenovirus serotype 5 vaccine vectors.

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations will likely limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. Ad5-specific neutralizing antibodies (NAbs) are thought to contribute substantially to anti-Ad5 immunity, but the potential importance of Ad5-specific T lymphocytes in this setting has not been fully characterized. Here we assess the relative contributions of Ad5-specific humoral and cellular immune responses in blunting the immunogenicity of a rAd5-Env vaccine in mice. Adoptive transfer of Ad5-specific NAbs resulted in a dramatic abrogation of Env-specific immune responses following immunization with rAd5-Env. Interestingly, adoptive transfer of Ad5-specific CD8(+) T lymphocytes also resulted in a significant and durable suppression of rAd5-Env immunogenicity. These data demonstrate that NAbs and CD8(+) T lymphocytes both contribute to immunity to Ad5. Novel adenovirus vectors that are currently being developed to circumvent the problem of preexisting anti-Ad5 immunity should therefore be designed to evade both humoral and cellular Ad5-specific immune responses.

Plasmid chemokines and colony-stimulating factors enhance the immunogenicity of DNA priming-viral vector boosting human immunodeficiency virus type 1 vaccines.

Heterologous "prime-boost" regimens that involve priming with plasmid DNA vaccines and boosting with recombinant viral vectors have been shown to elicit potent virus-specific cytotoxic T-lymphocyte responses. Increasing evidence, however, suggests that the utility of recombinant viral vectors in human populations will be significantly limited by preexisting antivector immunity. Here we demonstrate that the coadministration of plasmid chemokines and colony-stimulating factors with plasmid DNA vaccines markedly increases the immunogenicity of DNA prime-recombinant adenovirus serotype 5 (rAd5) boost and DNA prime-recombinant vaccinia virus (rVac) boost vaccine regimens in BALB/c mice. In mice with preexisting anti-Ad5 immunity, priming with the DNA vaccine alone followed by rAd5 boosting elicited only marginal immune responses. In contrast, cytokine-augmented DNA vaccine priming followed by rAd5 vector boosting was able to generate potent immune responses in mice with preexisting anti-Ad5 immunity. These data demonstrate that plasmid cytokines can markedly improve the immunogenicity of DNA prime-viral vector boost vaccine strategies and can partially compensate for antivector immunity.

Efficacy of superactivated charcoal administered late (3 hours) after acetaminophen overdose.

The purpose of this study was to investigate the effect of superactivated charcoal (SAC) given late after a drug overdose. Acetaminophen was chosen as our overdose drug because it has relatively few side effects, serum levels are easily attainable and measurable, and it is generally a common drug overdose. Forty-six healthy adult volunteers participated in this randomized, controlled study. Acetaminophen was administered the morning after an overnight fast. Thirteen participants received 2000 mg acetaminophen and the remaining 33 received 3000 mg. After 3 hours, half of the participants (22 of 46) received 75 g of SAC (Requa, Greenwich, CT) orally as a slurry in 8 oz of apple juice. Serum acetaminophen levels were measured at 4 and 7 hours after the initial acetaminophen administration. There were significantly lower uncorrected and corrected acetaminophen levels in the SAC group compared with the control group at both 4 and 7 hours after ingesting acetaminophen. This randomized human experimental design trial demonstrates some detoxification benefit in administering superactivated charcoal 3 hours after an overdose.

Acetaminophen levels 4 and 7 hours after 2000 and 3000 mg single doses in healthy adults.

PURPOSE: Therapeutic acetaminophen levels are not achieved at currently recommended doses. The purpose of this study is to determine acetaminophen levels in healthy adults after taking a single dose well in excess of the recommended dose. METHODS: 24 healthy adults received single 2 or 3 gram acetaminophen doses. Serum acetaminophen levels were drawn at 4 and 7 hours after the dose. RESULTS: The 2 gram doses (6 subjects) ranged from 23 to 40 mg/kg of body weight. The 3 gram single doses (18 subjects) ranged from 38 to 69 mg/kg of body weight. Mean 4-hour acetaminophen levels for 2 and 3 gram doses were 8.8 (SD 3.6, range 3-13) and 21.8 (SD 6.5, range 6-32) mcg/ml, respectively. Mean 7-hour acetaminophen levels for 2 and 3 gram doses were 1.5 (SD 1.4, range 0-3) and 7.7 (SD 4.6, range 0-17) mcg/ml, respectively. CONCLUSIONS: Dosing by weight (i.e., mg/kg) appears to provide a more predictable dose-response relationship. Optimal adult dosing appears to be somewhere in the 20 to 30 mg/kg range based on the premise that the 4 hour level is a trough level that should be in the low therapeutic range.

Adverse effects of superactivated charcoal administered to healthy volunteers.

OBJECTIVES: Activated charcoal is frequently administered to drug overdose patients, who may experience nausea and vomiting secondary to the drug overdose. Drinking a charcoal slurry orally may be difficult for them necessitating a gastric tube. The purpose of this study is to report the frequency of adverse effects from oral superactivated charcoal (SAC) given to healthy volunteers. METHODS: Healthy adult study subject volunteers were given a single 2000 mg (first 13 subjects) or 3000 mg (remaining 35 subjects) dose of acetaminophen. Subjects were randomized to receive no charcoal (ctrl) or 75 grams of SAC administered orally in a slurry 3 hours following the acetaminophen dose. The adverse effects of both groups were recorded and compared. RESULTS: There were 48 study subject runs. The mean age was 27.4 years (SD6.5). SAC was administered to 24 subjects. Adverse effect rates were as follows (*one-tail p < 0.05): black stool* (SAC 22/ 24, ctrl 0), constipation or abdominal fullness* (SAC 12/24, ctrl 0), nausea* (SAC 5/24, ctrl 0), vomiting (SAC 2/24, ctrl 0), diarrhea (SAC 2/24, ctrl 0), anal irritation (SAC 2/24, ctrl 0), drowsiness/fatigue (SAC 2/24, ctrl 3/24), dizziness/lightheadedness (SAC 0, ctrl 1/24), headache (SAC 4/24, ctrl 0). 7 of 24 SAC subjects and 20 of 24 ctrl subjects experienced no adverse effects at all* (other than black stools for the SAC subjects). Acetaminophen may have been blunted some adverse effects. Two SAC subjects could not finish the charcoal. For the 22 subjects who finished the charcoal, SAC consumption took a mean of 10.9 minutes (SD 11.8, range 1 to 50 minutes). Thirteen subjects finished it in 7 minutes or less. Six subjects took 19 minutes or longer to finish it. The 12 heavier subjects (> 71kg) completed SAC consumption significantly faster than the 12 lighter subjects (18.7 vs 7.8 minutes, p = 0.04, single sided). This comparison included the two subjects (both lighter) who did not finish SAC consumption, so this difference was no longer significant when these two subjects were removed. CONCLUSIONS: Superactivated charcoal consumption is associated with significant adverse effects in some healthy volunteers, which may impede a drug overdose patient's ability to willingly drink charcoal slurry in a reasonable period of time.