Randomized evaluation of fibrinogen vs placebo in complex cardiovascular surgery (REPLACE): a double-blind phase III study of haemostatic therapy.

BACKGROUND: Single-dose human fibrinogen concentrate (FCH) might have haemostatic benefits in complex cardiovascular surgery. METHODS: Patients undergoing elective aortic surgery requiring cardiopulmonary bypass were randomly assigned to receive FCH or placebo. Study medication was administered to patients with a 5 min bleeding mass of 60-250 g after separation from bypass and surgical haemostasis. A standardized algorithm for allogeneic blood product transfusion was followed if bleeding continued after study medication. RESULTS: 519 patients from 34 centres were randomized, of whom 152 (29%) met inclusion criteria for study medication. Median (IQR) pretreatment 5 min bleeding mass was 107 (76-138) and 91 (71-112) g in the FCH and placebo groups, respectively (P=0.13). More allogeneic blood product units were administered during the first 24 h after FCH, 5.0 (2.0-11.0), when compared with placebo, 3.0 (0.0-7.0), P=0.026. Fewer patients avoided transfusion in the FCH group (15.4%) compared with placebo (28.4%), P=0.047. The FCH immediately increased plasma fibrinogen concentration and fibrin-based clot strength. Adverse event rates were comparable in each group. CONCLUSIONS: Human fibrinogen concentrate was associated with increased allogeneic blood product transfusion, an unexpected finding contrary to previous studies. Human fibrinogen concentrate may not be effective in this setting when administered according to 5-minute bleeding mass. Low bleeding rates and normal-range plasma fibrinogen concentrations before study medication, and variability in adherence to the complex transfusion algorithm, may have contributed to these results. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier no. NCT01475669; EudraCT trial no. 2011-002685-20.

Validation and long term performance characteristics of a quantitative enzyme linked immunosorbent assay (ELISA) for human anti-PA IgG.

Accurate, reliable and standardized quantification of anti-protective antigen (PA) IgG antibody levels is essential for comparative analyses of anti-toxin immune responses in anthrax cases, recipients of PA-based anthrax vaccines and for evaluation of anti-PA based immunotherapies. We have previously reported the early performance characteristics and application of a quantitative anti-PA IgG enzyme linked immunosorbent assay. The principal application of this assay was in a Phase 4 human clinical trial of anthrax vaccine adsorbed (AVA, BioThrax), the central component of the CDC Anthrax Vaccine Research Program (AVRP) and in humans following bioterrorism associated Bacillus anthracis infection (Quinn et al., 2002; Quinn et al., 2004; Marano et al., 2008). The objective of the AVRP was to determine the feasibility of reducing the number of priming series and booster doses of the licensed Anthrax Vaccine Adsorbed (AVA) (BioThrax®; Emergent BioSolutions, Lansing, MI) and changing the route of administration from subcutaneous (SC) to intramuscular (IM) (Marano et al., 2008). In this paper we report the validation and long term performance characteristics of the assay during its six year application in the AVRP (2002-2008). The critical features are 1) extensive validation of the assay using two standard reference sera; 2) long term stability and 3) consistency of the data for quantitative analysis of human long term anti-PA IgG responses. The reportable value (RV) of the assay was expressed as anti-PA IgG concentration (µg/ml). Accuracy of the assay was high with a percent error (%ER) range of 1.6-11.4%. Overall intra-operator and intermediate precision were high with Coefficients of Variation (%CVs) of 2.5-15.4% and 6.3-13.2%, respectively. The assay demonstrated excellent dilutional linearity for human sera using log(10) transformed data with the slope=0.95 to 0.99, intercept=0.02 to 0.06 and r(2)=0.980-0.987. The assay was robust, tolerating changes in serum incubation temperatures from 35 to 39°C, serum incubation times from 55 to 65min and changes in key reagents. The long-term assay stability over 6years using consecutive reference sera AVR414 and AVR801 demonstrated sustained high accuracy and precision for the assay, confirming its suitability for long term studies of PA protein-based anthrax vaccines.

Analysis of anti-protective antigen IgG subclass distribution in recipients of anthrax vaccine adsorbed (AVA) and patients with cutaneous and inhalation anthrax.

The anti-PA IgG1, IgG2, IgG3, and IgG4 subclass responses to clinical anthrax and to different numbers of anthrax vaccine adsorbed (AVA, BioThrax) injections were determined in a cross-sectional study of sera from 63 vaccinees and 13 clinical anthrax patients. The data show that both vaccination with three AVA injections and clinical anthrax elicit anti-PA IgG1, IgG2, and IgG3 subclass responses. An anti-PA IgG4 response was detected in AVA recipients after the fourth injection. The anthrax lethal toxin (LTx) neutralization efficacy of sera from recipients who received 4 to > or =10 AVA injections did not vary significantly in relation to changes in distribution of anti-PA IgG1 and IgG4 subclasses.

Mass value assignment of total and subclass immunoglobulin G in a human standard anthrax reference serum.

An anti-Anthrax Vaccine Adsorbed (anti-AVA) standard human reference serum pool, AVR414, has been prepared, and the total and protective antigen (PA)-specific immunoglobulin G (IgG) were quantified. AVR414 was prepared by plasmapheresis of healthy adults who had received a minimum of four subcutaneous injections of AVA. Mass values (in milligrams per milliliter) for total IgG and IgG subclasses 1 to 4 were determined by radial immunodiffusion. Anti-PA-specific IgG assignment (in micrograms per milliliter) was done by consensus of two complementary approaches: homologous enzyme-linked immunosorbent assay (ELISA) with affinity-purified anti-PA IgG as a calibrator and summation of mean PA-specific IgG subclass concentrations determined by IgG subclass-specific ELISA using the United States National Reference Preparation for Human Serum Proteins as a standard. The total IgG concentration assigned to AVR414 reference serum was 8.33 mg/ml. IgG subclass concentrations were the following: for IgG1, 4.48 mg/ml; for IgG2, 3.35 mg/ml; for IgG3, 0.37 mg/ml; and for IgG4, 0.30 mg/ml. The assigned mass value for total anti-PA-specific IgG was 141.2 microg/ml. Anti-PA-specific IgG subclass concentrations were the following: for IgG1, 79.6 microg/ml; for IgG2, 35.3 microg/ml; for IgG3, 3.2 microg/ml; and for IgG4, 25.3 microg/ml. Human reference serum pool AVR414 will have direct application in the standardization of anthrax serological assays, in reagent qualification, and as a standard for quantification of PA-specific IgG in humans who have been vaccinated with or otherwise exposed to Bacillus anthracis PA.

Assessment of chemoreflex sensitivity in free breathing young subjects by correction for respiratory influence.

BACKGROUND: The assessment of autonomic function is an important tool for risk stratification in critically ill patients. Peripheral cardiac chemoreflex sensitivity has been considered a marker for increased risk of sudden cardiac death. In normals, the evaluation of peripheral cardiac chemoreflex sensitivity is performed under controlled breathing conditions during inhalation of hypoxic gas. Since this is poorly tolerated by patients, they are commonly studied under hyperoxic conditions, which are not physiological. METHODS: We studied 20 healthy volunteers who underwent free and controlled breathing of a hypoxic gas mixture (10% O2 in N2) over 5 min. Values of peripheral cardiac chemoreflex sensitivity, corrected for respiratory influence, were compared with the results obtained experimentally under controlled breathing conditions in the same subjects. RESULTS: We found a substantial difference between values obtained during free and controlled breathing (3.64 +/- 0.81 vs. 1.53 +/- 0.32 ms/mmHg, respectively; P < 0.05). After application of a respiratory correction, described and validated in this article, no significant difference was seen for these values (0.89 +/-0.91 vs. 1.53 +/- 0.32 ms/mmHg, P = 0.46). CONCLUSIONS: This approach allows the evaluation of peripheral cardiac chemoreflex sensitivity in free breathing subjects. This correction could improve the assessment of cardiac chemoreflex sensitivity in patients with cardiorespiratory disorders, who find it difficult to control their breathing according to an experimental protocol.

Adolescents with ASCUS: are they a high risk group?

Population demographics, risk behaviors, and compliance rates for the management of an ASCUS (atypical squamous cells of undetermined significance) diagnosis are not well studied in the adolescent population. From June 1994 to December 1996, 1,175 Papanicalou (pap) smears were performed in an urban adolescent clinic on patients age 12 to 18. Of these, 124 (10.5%) were diagnosed with ASCUS or ASCUS with a qualifying statement. A retrospective chart review (n=83) and telephone interview was performed on patients with ASCUS. Ninety-nine percent of enrollees were African American. Comparisons were made between those patients with normal pap smears and those with ASCUS. No statistically significant difference existed pertaining to age at pap smear, age at menarche, age at first coitus, and education level. A positive association was found in the ASCUS group for the presence of sexually transmitted diseases (P < 0.001), number of sexual partners (P < 0.0007), and pregnancy (P < 0.001). Of the 80% of patients who had an ASCUS diagnosis and were referred for colposcopy (n = 62), only 61% attended their appointment (n = 38). Thirty-nine percent of these patients were aware of an abnormal diagnosis after colposcopy. For those that attended colposcopy, 56% were accompanied by a parent. For those who were not compliant with attendance, none cited parental consent for the procedure as a barrier to obtaining treatment. Adolescent females in an urban setting with multiple sexual partners, history of sexually transmitted diseases, and prior pregnancy are at a greater risk for ASCUS on cervicovaginal smear when compared to their age-matched controls. In addition, the adolescent compliance rate for colposcopy is low. We, therefore, recommend that these adolescent females be observed diligently.

The CD44 variant isoforms CD44v6 and CD44v7 are expressed by distinct leukocyte subpopulations and exert non-overlapping functional activities.

We have described recently that anti-CD44s, anti-CD44v6 and anti-CD44v7 interfere with delayed-type hypersensitivity (DTH) reactions. Yet, TNBS-induced colitis can be cured only by anti-CD44v7. To clarify the mechanisms underlying the divergent functional activities of CD44v6 and CD44v7 we explored their contribution to lymphocyte activation in vivo and in vitro. CD44v6 and CD44v7 are distinctly expressed on subpopulations of activated lymphocytes. Expression of CD44v6 is mainly restricted to T cell blasts. CD44v7 has been detected on CD4(+) cells, B cells and monocytes. Mitogenic and antigenic stimulation of lymphocytes in vitro was impaired in the presence of anti-CD44v6 and anti-CD44v7. Accordingly, anti-CD44v6 and anti-CD44v7 mitigated the DTH reaction in 2,4-dinitro-1-fluorobenzene-sensitized and challenged mice. However, the seemingly similar effects of CD44v6- and CD44v7-specific antibodies resulted from different activities. Anti-CD44v6 treatment led to a down-regulation of IL-2 and IFN-gamma production predominantly by CD8(+) cells. In anti-CD44v7-treated mice expression of IL-12 was decreased. Elevated levels of IL-10 accompanied this reduction. The latter resulted from an anti-CD44v7-mediated blockade of interactions between CD4(+) cells and monocytes as well as an active triggering of B cells. Thus, anti-CD44v6 and anti-CD44v7 interfere with lymphocyte activation at very specific points. CD44v6 functions predominantly at the T cell level. CD44v7 influences production of proinflammatory cytokines by B cells as well as an interaction between CD4(+) cells and antigen-presenting cells. As CD44 isoforms do not differ in their intracytoplasmatic tail, the distinct activities must result from expression on different leukocyte subsets and interactions with distinct ligands.

CD44 variant isoforms on blood leukocytes in chronic inflammatory bowel disease and other systemic autoimmune diseases.

We have described recently that TNBS-induced colitis, an animal model of chronic inflammatory bowel disease (IBD), can be cured by treatment with anti-CD44v7. This finding led us to evaluate whether CD44v7 may be of functional importance in patients with IBD. Expression of CD44 variant isoforms (CD44v) has been evaluated on PBMC of 46 patients with IBD, 43 patients with autoimmune diseases not affecting the gastrointestinal tract, 26 patients with nonautoimmune disease, and 24 healthy donors. In all groups, expression of CD44v on freshly harvested PBMC was not above or was borderline above background levels. After in vitro stimulation, expression of CD44 standard (CD44s) and CD44v6 was strongly up-regulated. Exclusively on PBMC of patients with autoimmune disease, high expression of CD44v3 and CD44v7 was observed. CD44v3 and CD44v7 were mainly expressed on subsets of CD4+ lymphocytes, B cells, and monocytes; CD44v6 was predominantly detected on CD4+ and CD8+ cells. Considering functional activity, CD44v7 apparently exerted a dual effect. After culturing PBMC in the presence of anti-CD44v7, a higher percentage of cells produced IL-10. This was irrespective of whether the PBMC were derived from healthy donors or from patients with autoimmune disease or IBD. On the other hand, PBMC of all donors proliferated upon cross-linking of CD3 and CD44s or CD3 and CD44v6. Instead, costimulatory activity of CD44v7 was seen only in PBMC of patients with autoimmune disease and IBD. Because expression and function of CD44v7 in patients with systemic autoimmune disease and IBD have been much like the ones in mice suffering of TNBS-induced colitis, it is tempting to speculate that blockade of CD44v7 could also be of therapeutic relevance in the human diseases.

Dissemination of lymphocytic choriomeningitis virus from the gastric mucosa requires G protein-coupled signaling.

The gastric mucosa is an important portal of entry for lymphocytic choriomeningitis virus (LCMV) infections. Within hours after intragastric (i.g.) inoculation, virus appears in the gastric epithelia, then in the mesenteric lymph nodes and spleen, and then in the liver and brain. By 72 h i.g.-inoculated virus is widely disseminated and equivalent to intravenous (i.v.) infection (S. K. Rai, B. K. Micales, M. S. Wu, D. S. Cheung, T. D. Pugh, G. E. Lyons, and M. S. Salvato. Am. J. Pathol. 151:633-639, 1997). Pretreatment of mice with a G protein inhibitor, pertussis toxin (PTx), delays LCMV dissemination after i.g., but not after i.v., inoculation. Delayed infection was confirmed by plaque assays, by reverse transcription-PCR, and by in situ hybridization. The differential PTx effect on i.v. and i.g. infections indicates that dissemination from the gastric mucosa requires signals transduced through heterotrimeric G protein complexes. PTx has no direct effect on LCMV replication, but it modulates integrin expression in part by blocking chemokine signals. LCMV infection of macrophages up-regulates CD11a, and PTx treatment counteracts this. PTx may prevent early LCMV dissemination by inhibiting the G protein-coupled chemotactic response of macrophages infected during the initial exposure, thus blocking systemic virus spread.

Effects of oil-soluble organosulfur compounds from garlic on doxorubicin-induced lipid peroxidation.

Clinical efficacy of doxorubicin is compromised due to free radical generation leading to cardiac toxicity. Oil-soluble organosulfur compounds, diallyl sulfide (DAS), diallyl disulfide (DADS), dipropyl sulfide (DPS) and dipropyl disulfide (DPDS), present in garlic were examined for their antiperoxidant effects. DADS inhibited liver microsomal lipid peroxidation induced by NADPH, ascorbate and doxorubicin. DAS, DPS and DPDS were ineffective inhibitors of liver microsomal lipid peroxidation. DADS could be used in combination with doxorubicin to protect oxidative injuries to improve the clinical efficacy of doxorubicin.

Anti-glycoprotein B monoclonal antibody protects T cell-depleted mice against herpes simplex virus infection by inhibition of virus replication at the inoculated mucous membranes.

A monoclonal antibody (MAb 2c) specific for glycoprotein B of herpes simplex virus (HSV) mediated clearance of the virus from the infected mucous membranes. Young adult C57BL/6J mice were inoculated intravaginally with HSV type 1 and injected intraperitoneally either 24 and 72 h or 65 and 265 h post-inoculation with a polyclonal immune serum or the MAb 2c, both adjusted to the same neutralizing capacity. Immunization with the polyclonal immune serum did not alter the duration of virus shedding from the genital mucous membranes although a lethal outcome of infection was clearly prevented. Immunization with the MAb, however, resulted in a rapid clearance of the virus from the genital tract thus completely inhibiting genital inflammation and lethality. The same effects were achieved in mice depleted in vivo of CD4+ T cells although peripheral virus replication continued longer in these mice. In mice depleted of both CD4+ and CD8+ T cells the polyclonal immune serum was no longer able to protect against lethality, and virus replication in the mucous membranes persisted until the mice died. In contrast, after treatment with the MAb peripheral infection was quickly eliminated and all mice survived. These findings indicate that clearance of the virus from the primary site of replication can be mediated by humoral immunity. The relevance of this observation for vaccination against HSV is discussed.

The role of the immune system in establishment of herpes simplex virus latency--studies using CD4+ T-cell depleted mice.

The immunological mechanisms involved in establishment of herpes simplex virus (HSV) latency were studied in normal and CD4+ T-cell depleted C57BL/6J mice following intravaginal infection. During transition from acute to latent ganglionic infection two consecutive processes were observed: first, clearance of infectious virus from the ganglia, and second, reduction of the number of infected ganglia.

The pathology of the eye in armadillos experimentally infected with Mycobacterium leprae.

One hundred and twenty-seven eyes from 66 Mycobacterium leprae inoculated armadillos were studied histologically and some ultrastructurally. Inflammatory reactions were found in the following extraocular tissues: the eyelid, including the orbicularis muscle and the third eyelid, extraocular muscles, tear gland and Harder's gland. The early and slight changes of the intraocular tissues, small amounts of lymphocytes, plasma cells and macrophage infiltrations were confined to the area around the anterior angle specifically within the trabeculae and the adjacent ciliary body, the root of the iris and the limbus region of the cornea. But in the cases with severe lesions the whole uvea was densely infiltrated with large, foamy macrophages intermingled with small amounts of lymphocytes, plasma cells and frequently, neutrophils. No specific necrosis of the granulomas was seen. No explanation for the neutrophil infiltrations was given. The lesions in the cornea were significantly less severe than those in the uvea. Retinal lesions comprised of macrophage infiltrations were all obvious extensions of the adjacent uvea lesions. Acid-fast bacilla (AFB) were found within all tissues. The infection of the intraocular tissues in the armadillo eyes seemed to be mainly, if not solely, haematogenous.

Control of the tRNA-tufB operon in Escherichia coli. 1. rRNA gene dosage effects and growth-rate-dependent regulation.

'Ribosome feedback' effects on the expression of the genes specifying tRNA and EF-Tu in E. coli have been studied at increased rrnB doses (rRNA gene doses). We confirm previous observations that the introduction into the cell of a multicopy plasmid carrying the rrnB operon reduces the cellular content of most tRNAs, including those encoded by the tRNA-tufB operon, but leaves the 5S rRNA content unaffected. Increase of the dosage of intact, but not of deleted rRNA genes, causes a slight drop in total EF-Tu that can be fully accounted for by a decrease in EF-TuB level. The drop in EF-TuB content (approx. 25%) is much smaller than that in tRNA content (approx. 80%). The synthesis rate of total EF-Tu is hardly affected, indicating that the turnover of EF-Tu has not changed. The ratio of tRNA over tuf RNA synthesis rates remains the same after elevation of rrnB dosage. Considering the large decrease in tRNA content this means that both RNA synthesis rates decrease to approximately the same extent. The relatively small drop in EF-Tu synthesis must be due, therefore, to an enhancement of the number of EF-Tu molecules synthesized per mRNA molecule. Apparently a post-transcriptional mechanism, regulating EF-Tu synthesis, becomes operative under these conditions. Growth-rate-dependent regulation of the tRNA-tufB operon has been studied using lysogens carrying tRNA':lacZ and tRNA-tufB':lacZ operon fusions and a tufB':lacZ' gene fusion. These experiments show that the cellular contents of tRNA, tufB RNA and EF-TuB vary in direct proportion to the growth rate. This indicates that growth rate control of tRNA-tufB operon transcription resembles that of stable RNA operons and not of r-protein operons, and that the read-through of the terminator at the end of the tRNA gene cluster remains unaltered.

Control of the tRNA-tufB operon in Escherichia coli. 2. Mechanisms of the feedback inhibition of tufB expression studied in vivo and in vitro.

The mechanism underlying feedback inhibition of tufB expression has been studied in vivo by gene-dosage experiments and by gene and operon fusions involving lacZ. Raising the cellular EF-Tu content, by introducing a multicopy plasmid encoding EF-TuA into the cell, repressed the level of EF-TuB but left the content of tRNA(Thr)3, encoded by the tRNA-tufB operon, unaffected. This indicates that autoregulation of chromosomal tufB expression does not occur by modulating transcription initiation at the promoter of the tRNA-tufB operon. This conclusion is further substantiated by experiments with a tRNA':lacZ operon fusion. The molecular ratio of chromosome-borne tufA and tufB transcripts also remained unaltered under conditions of excess EF-Tu, though experiments with a tRNA-tufB':lacZ operon fusion showed a decrease of tufB transcripts. Our data further exclude drastic effects of the autogenous repressor on processing of the contranscript of the operon into monocistronic tufB RNA and on alteration of EF-TuB turnover. Two possible mechanisms remain, which cannot yet be decided between. One is modulation of EF-Tu by transcription termination either directly or indirectly by affecting antitermination. The second is translational repression. In vitro translation of transcripts derived from SP6 clones did not reveal any feedback inhibition of EF-TuB synthesis. Surprisingly, addition of EF-Tu to a coupled transcription/translation systems was found to block transcription initiation at the primary promoter of the tRNA-tufB operon by over 90%. Although this in vitro effect of EF-Tu could not be demonstrated in vivo, possibly because of a difference in higher-order structure between plasmid-borne and chromosome-borne DNA, it indicates that under certain conditions EF-Tu binds very specifically to the tRNA-tufB operon promoter or its upstream region.

Transcription of the tRNA-tufB operon of Escherichia coli: activation, termination and antitermination.

Signals setting the level of transcription of the tRNA-tufB operon have been studied by deletion mapping. TufB transcription was measured in vivo with plasmid-borne tRNA-tufB:galk operon fusions. Removal of the sequences from -133 to -58 with respect to the transcription start point, results in a 90% decrease of tufB transcription. This demonstrates the presence of a region, upstream of the tRNA-tufB promoter, that enhances the expression of the operon. DNA fragments bearing this upstream activator region do not display an abnormal electrophoretic mobility, as has been observed for the rrnB P1 upstream activator. Deletions starting in the first tRNA gene and directing towards tufB reveal at least two sites that influence tufB transcription. One signals transcription termination in the intergenic region between thrT and tufB. The other may be involved in antitermination. Possible mechanisms underlying antitermination and termination are considered in the light of the nucleotide sequence.

The tRNA-tufB operon transcription termination and processing upstream from tufB.

Two genes, tufA and tufB, located at 73 and 88 minutes of the Escherichia coli linkage map, code for the polypeptide chain elongation factor EF-Tu. tufB is transcribed with four upstream tRNA genes, thrU, tyrU, glyT and thrT, into a cotranscript of approximately 1800 nucleotides. Here we show that in vivo processing yields a 1320 nucleotide transcript of tufB. S1 nuclease fine mapping reveals that the processing site is located in the intergenic region at about 72 to 74 nucleotides upstream from the initiation codon of the tufB cistron. A deletion in the cloned tRNA-tufB operon, encompassing the 3' half of thrU, the complete tyrU, glyT, thrT genes and ten nucleotides of the intergenic region, causes a threefold increase of the rate of plasmid tufB transcription, a fourfold increase of plasmid-borne tufB RNA and a twofold increase of plasmid-borne EF-TuB. We conclude that the deletion has eliminated a transcription termination site probably located after the thrT gene. Termination at this site uncouples tRNA synthesis from tufB transcription.