Survival analysis and microarray profiling identify Cd40 as a candidate for the Salmonella susceptibility locus, Ity5.

The outcome of infection with Salmonella Typhimurium in mouse models of human typhoid fever is dependent upon a coordinated complex immune response. A panel of recombinant congenic strains (RCS) derived from reciprocal backcross of A/J and C57BL/6J mice was screened for their susceptibility to Salmonella infection and two susceptibility loci, Ity4 (Immunity to Typhimurium locus 4) and Ity5, were identified. We validated Ity5 in a genetic environment free of the impact of Ity4 using a cross between A/J and 129S6. Using a time-series analysis of genome-wide transcription during infection, comparing A/J with AcB60 mice having a C57BL/6J-derived Ity5 interval, we have identified the differential expression of the positional candidate gene Cd40, Cd40-associated signaling pathways, and the differential expression of numerous genes expressed in neutrophils. CD40 is known to coordinate T cell-dependent B-cell responses and myeloid cell activation. In fact, CD40 signaling is altered in A/J mice as seen by impaired IgM upregulation during infection, decreased Ig class switching, neutropenia, reduced granulocyte recruitment in response to infection and inflammation, and decreased ERK1/2 activity. These results suggest that altered CD40 signaling and granulocyte recruitment in response to infection are responsible for the Ity5-associated Salmonella susceptibility of A/J mice.

Architecture of the RNA polymerase II-Mediator core initiation complex.

The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

Impact of Usp18 and IFN signaling in Salmonella-induced typhlitis.

In humans, Salmonella infection causes two major clinical diseases, typhoid fever and a self-limiting gastro-enteritidis. Salmonella transmission occurs by the fecal-oral route and the interactions between the bacteria and the digestive tract epithelium are central to the outcome of the infection. Using a mouse model of typhoid fever, we previously identified a mutation in USP18 affecting type I interferon (IFN) signaling resulting in increased susceptibility to systemic Salmonella infection. In this study, we demonstrate the effects of this mutation during the early response to Salmonella using a model of typhlitis. Mutant Usp18 mice showed a minimal inflammatory response early after Salmonella Typhimurium infection that was associated with low pathologic scores and low IFN-γ production. This resulted in an increased interaction of Salmonella with the cecal epithelium and earlier systemic dissemination of the bacteria. The global transcriptional signature in the cecum of mouse during Salmonella infection showed normal expression of tissue specific genes and upregulation of type I IFN pathway in mutant mice. In control mice, there was a significant over-representation of genes involved in cellular recruitment and antibacterial activity paralleling the histopathological features. These results show the impact of USP18 in the development of Salmonella-induced typhlitis.

Incremental expression of Tlr4 correlates with mouse resistance to Salmonella infection and fine regulation of relevant immune genes.

The mouse response to Salmonella Typhimurium infection is partly controlled through detection of the bacterium lipopolysaccharide by the host pattern recognition receptor, Toll-like receptor 4 (Tlr4). Mice deficient in Tlr4 signaling are extremely susceptible to Salmonella infection with a 1,000-fold reduction in LD(50). In a previous study, we showed, using transgenic mice carrying one, three, six and >30 copies of Tlr4, that the level of expression of this gene influences the outcome of Salmonella infection, with a plateau effect starting at three copies. In the present study, we further investigate the impact of Tlr4 during Salmonella infection in mice expressing Tlr4 at slightly sub-normal, normal and slightly supra-normal levels by comparing host responses in mice carrying one, two and three copies of Tlr4 on the same genetic background. We describe in detail the in vivo host response to pathogenic Salmonella and show for the first time, in this narrow range of Tlr4 expression, an incremental protective effect against Salmonella due to improved control of bacterial growth in target organs and increased expression of important immune response genes in the spleen.

High resolution crystal structures of T4 phage beta-glucosyltransferase: induced fit and effect of substrate and metal binding.

beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 that transfers glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine bases of phage T4 DNA. We report six X-ray structures of the substrate-free and the UDP-bound enzyme. Four also contain metal ions which activate the enzyme, including Mg(2+) in forms 1 and 2 and Mn(2+) or Ca(2+). The substrate-free BGT structure differs by a domain movement from one previously determined in another space group. Further domain movements are seen in the complex with UDP and the four UDP-metal complexes. Mg(2+), Mn(2+) and Ca(2+) bind near the beta-phosphate of the nucleotide, but they occupy slightly different positions and have different ligands depending on the metal and the crystal form. Whilst the metal site observed in these complexes with the product UDP is not compatible with a role in activating glucose transfer, it approximates the position of the positive charge in the oxocarbonium ion thought to form on the glucose moiety of the substrate during catalysis.

LPS-hyporesponsiveness of mnd mice is associated with a mutation in Toll-like receptor 4.

Toll-like receptors (Tlrs) are transmembrane proteins that have recently been shown to play a critical role in the innate immune recognition of microbial constituents. Among this family, Tlr4 is a crucial signal transducer for lipopolysaccharide (LPS), the major component of the Gram-negative bacteria outer cell membrane. In this paper, we report that C57BL/6.KB2-mnd mice, a model of neuronal ceroid lipofuscinosis, do not respond to LPS. This defect is associated with a spontaneous mutation in Tlr4 consisting of a large insertion within exon 2 predicting a frameshift mutation and a truncated protein.

Cloning and characterization of the murine toll-like receptor 5 (Tlr5) gene: sequence and mRNA expression studies in Salmonella-susceptible MOLF/Ei mice.

Toll-like receptors (TLRs) are a group of evolutionarily conserved pattern recognition receptors involved in the activation of the immune system in response to various pathogens. In this paper, we describe the cloning and characterization of the mouse homologue of human TLR5. Mouse Tlr5 encodes a 859-amino-acid protein that contains an N-terminal signal sequence, a leucine-rich repeat extracellular domain, a short transmembrane domain typical of type I transmembrane proteins, and a Toll/interleukin-1R signaling domain characteristic of all TLR proteins. The mouse Tlr5 protein shows 81% homology to human TLR5 and approximately 40% similarity to other TLR family members. Northern blot analysis reveals that Tlr5 is expressed predominantly in liver and lung with low-level expression in most other tissues examined. We have mapped Tlr5 to distal chromosome 1 using the (C57BL/6J x Mus spretus) x C57BL/6J Jackson BSB panel as well as a (C57BL/6J x MOLF/Ei)F(2) panel with the following position: D1Mit112-8.0 cM-Tlr5-9.6 cM-D1Mit17. The presence of a quantitative trait locus for susceptibility to Salmonella typhimurium on distal chromosome 1 prompted the examination of Tlr5 in susceptible MOLF/Ei mice. Polymorphic sequence variants in Tlr5 allowed us to identify a unique 4-allele haplotype in MOLF/Ei. Furthermore, using both Northern blot analysis and reverse transcription-polymerase chain reaction, we have shown a reduced expression of Tlr5 during infection of MOLF/Ei mice with Salmonella. The assignment of Tlr5 to a chromosomal region known to harbor a Salmonella-susceptibility locus together with decreased expression of Tlr5 mRNA in liver of susceptible MOLF/Ei mice suggests the possibility that, as with other members of this family, Tlr5 may play a role in host response to bacterial gram-negative infections.

Cutting edge: functional characterization of the effect of the C3H/HeJ defect in mice that lack an Lpsn gene: in vivo evidence for a dominant negative mutation.

A point mutation in the Tlr4 gene, which encodes Toll-like receptor 4, has recently been proposed to underlie LPS hyporesponsiveness in C3H/HeJ mice (Lpsd). The data presented herein demonstrate that F1 progeny from crosses between mice that carry a approximately 9-cM deletion of chromosome 4 (including deletion of LpsTlr4) and C3H/HeJ mice (i.e., Lps0 x Lpsd F1 mice) exhibit a pattern of LPS sensitivity, measured by TNF activity, that is indistinguishable from that exhibited by Lpsn x Lpsd F1 progeny and whose average response is "intermediate" to parental responses. Thus, these data provide clear functional support for the hypothesis that the C3H/HeJ defect exerts a dominant negative effect on LPS sensitivity; however, expression of a normal Toll-like receptor 4 molecule is apparently not required.

Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4)

Bacterial lipopolysaccharide (LPS) provokes a vigorous, generalized proinflammatory state in the infected host. Genetic regulation of this response has been localized to the Lps locus on mouse chromosome 4, through study of the C3H/HeJ and C57BL/10ScCr inbred strains. Both C3H/HeJ and C57BL/10ScCr mice are homozygous for a mutant Lps allele (Lpsd/d) that confers hyporesponsiveness to LPS challenge, and therefore exhibit natural tolerance to its lethal effects. Genetic and physical mapping of 1,345 backcross progeny segregating this mutant phenotype confined Lps to a 0.9-cM interval spanning 1.7 Mb. Three transcription units were identified within the candidate interval, including Toll-like receptor 4 (Tlr4), part of a protein family with members that have been implicated in LPS-induced cell signaling. C3H/HeJ mice have a point mutation within the coding region of the Tlr4 gene, resulting in a nonconservative substitution of a highly conserved proline by histidine at codon 712, whereas C57BL/ 10ScCr mice exhibit a deletion of Tlr4. Identification of distinct mutations involving the same gene at the Lps locus in two different hyporesponsive inbred mouse strains strongly supports the hypothesis that altered Tlr4 function is responsible for endotoxin tolerance.

A randomized, placebo-controlled, double-blind trial of mesalamine in the maintenance of remission of Crohn's disease. The Canadian Mesalamine for Remission of Crohn's Disease Study Group.

BACKGROUND & AIMS: The efficacy of mesalamine for the maintenance of remission in patients with Crohn's disease is controversial. The aim of this study was to conduct a double-blind, placebo-controlled study of mesalamine (750 mg four times a day for 48 weeks) in maintaining remission in 293 patients with Crohn's disease. Patients were stratified according to the method of induction of remission (medical or surgical). METHODS: Patients were assessed at weeks 4, 12, 24, 36, and 48. Relapse was defined as a Crohn's Disease Activity Index of >150 (+60 points over baseline). RESULTS: Of the 293 patients, 246 (84%) returned for at least 4 weeks of follow-up and were included in the final analysis. Thirty of the 118 (25%) who received mesalamine had a relapse compared with 47 of 128 (36%) receiving placebo (P = 0.056). Among those with relapse, the time to relapse was 119 days for the mesalamine-treated patients compared with 109 days for placebo-treated patients (P = NS). However, 25% of mesalamine-treated patients had relapsed by 249 days of follow-up compared with 154 days for placebo-treated patients. Subgroup analysis showed that patients with ileocecal-colonic disease or patients who were women had fewer relapses on mesalamine therapy than placebo-treated patients (21% vs. 41%, P = 0.018; and 19% vs. 41%, P = 0.003, respectively). CONCLUSIONS: Mesalamine treatment reduced relapse compared with placebo treatment, although conventional statistical significance was not achieved.

A high-resolution map in the chromosomal region surrounding the Lps locus.

The Lps locus on mouse chromosome 4 controls host responsiveness to lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria. The C3H/HeJ inbred mouse strain is characterized by a mutant Lps allel (Lpsd) that renders it hyporesponsive to LPS and naturally tolerant of its lethal effects. To identify the Lps gene by a positional cloning strategy, we have generated a high-resolution linkage map of the chromosomal region surrounding this locus. We have analyzed a total of 1604 backcross mice from a preexisting interspecific backcross panel of 259 (Mus spretus x C57BL/6J)F1 x C57BL/6J and two novel panels of 597 (DBA/2J x C3H/HeJ)F1 x C3H/HeJ and 748 (C57BL/6J x C3H/HeJ)F1 x C3H/HeJ segregating at Lps. A total of 50 DNA markers have been mapped in a 11.8-cM span overlapping the Lps locus. This positions the Lps locus within a 1.1-cM interval, flanked proximally by a large cluster of markers, including three know genes (Cd301, Hxb, and Ambp), and distally by two microsatellite markers (D4Mit7/D4Mit178). The localization of the Lps locus is several centimorgans proximal to that previously assigned.

Antianginal efficacy and safety of controlled-delivery diltiazem QD versus an equivalent dose of immediate-release diltiazem TID.

Controlled-delivery once-daily diltiazem (qd), 180 mg and 360 mg, was assessed in two multicenter, randomized, double-blind, placebo-controlled trials using a 3 x 3 Latin square design. Both studies compared the controlled-delivery dosage form to the same total daily dose of immediate-release diltiazem administered three times daily (tid) and to placebo. The primary measure of efficacy was the time to termination of the exercise tolerance test (ETT) at 2, 8, and 24 hours after the morning dose. There were no significant differences in time to ETT termination between the qd and tid formulations at any time, except at 24 hours with 180 mg qd versus 60 mg tid. The comparison to placebo showed that diltiazem 180 mg qd, 360 mg qd, and 120 mg tid significantly lengthened the time to ETT termination (p < 0.05) at all time points, while diltiazem 60 mg tid did not differ from placebo at any time point. The qd formulation also increased the time to 1-mm ST-segment depression and reduced the number of angina attacks and the amount of nitroglycerin used when compared to placebo. No new or unusual adverse events were noted. Diltiazem controlled-release capsules administered once daily are safe and effective for the treatment of patients with chronic stable angina.

Three-month efficacy and safety of once-daily diltiazem in chronic stable angina pectoris.

The 3-month efficacy and safety of a once-daily controlled formulation of diltiazem (180 to 360 mg/day) were assessed in a study of 54 patients with angina pectoris. This multicenter study was a nonrandomized, placebo run-in, open-label, 3-month trial followed by a 1-week, double-blind, randomized period during which most patients (89%) received placebo. There were only minimal changes in the time to termination (mean change +/- SEM -5.8 +/- 9.6 seconds), time to onset of angina (10.5 +/- 12.2 seconds), and the time to 1 mm ST-segment depression (2.9 +/- 12.5 seconds) from the end of the titration phase to the end of the open-label study. There were, however, statistically significant differences between the end of the 3-month treatment phase and the end of the 1-week randomized placebo phase for those 3 efficacy parameters (-37.3 +/- 11.2, -58.6 +/- 13.6, and -45.6 +/- 16.4 seconds, respectively). Diltiazem significantly decreased the frequency of anginal attacks and nitroglycerin use at the end of the 3-month treatment phase compared with results at the end of the randomized double-blind placebo phase. No new or unusual adverse events were reported during treatment. The present results suggest that there is no loss of efficacy of once-a-day diltiazem when administered for a long period to patients with chronic stable angina pectoris.

Role of the beta-lactamase of Campylobacter jejuni in resistance to beta-lactam agents.

We studied the role of the beta-lactamase of Campylobacter jejuni in resistance to beta-lactam agents. beta-Lactamase-positive strains were more resistant than beta-lactamase-negative strains to amoxicillin, ampicillin, and ticarcillin (P less than 0.05). With penicillin G, piperacillin, imipenem, and six cephalosporins, the susceptibility levels were similar for both beta-lactamase-positive and -negative strains. By using spectrophotometric and microbiological assays, the beta-lactamase from three strains hydrolyzed ampicillin, amoxicillin, penicillin G, cloxacillin, and, partially, cephalothin. Ticarcillin and piperacillin were partially hydrolyzed in the microbiological assay. There was no activity against five other cephalosporins or imipenem. Isoelectric focusing of the enzyme showed a pI of 8.8. Tazobactam was the best inhibitor of the enzyme, followed by clavulanic acid, sulbactam, and cefoxitin, while EDTA and p-chloromercuribenzoate had no activity. All beta-lactamase-positive strains became susceptible to amoxicillin and ampicillin with 1 micrograms of clavulanic acid per ml. With the same inhibitor, there was a reduced but significant effect for ticarcillin but no effect for penicillin G or piperacillin. Sulbactam had no effect and tazobactam was effective only at 2 micrograms/ml on amoxicillin and ampicillin. The beta-lactamase of C. jejuni seems to be a penicillinase with a role in resistance for only amoxicillin, ampicillin, and ticarcillin.

Pharmacokinetics of intravenous diltiazem and five of its metabolites in patients with chronic renal failure and in healthy volunteers.

The pharmacokinetics of diltiazem were studied in seven patients with chronic renal failure (CRF) not requiring dialysis and in three healthy volunteers after a rapid i.v. infusion of 20 mg. Mean plasma concentrations at the end of infusion were 3.15 times higher in patients with CRF than in healthy volunteers. From 0.5 to 12 h post-infusion, the difference remained between 25 per cent and 73 per cent. Mean AUC0-infinity was statistically greater in patients than in volunteers while mean V area, CLtot, and CLren were statistically lower. The t1/2 alpha and t1/2 beta values were not significantly (p greater than 0.05) different between patients and volunteers. Renal excretion was statistically more important in volunteers (6.6 per cent of the dose) than in patients (1.2 per cent of the dose). We therefore conclude that CRF does not influence t1/2 beta of diltiazem but it interferes with the extent and possibly the rate of its extravascular distribution. That could result in transient high plasma concentrations after rapid i.v. infusion.

Pharmacokinetics of oral diltiazem and five of its metabolites in patients with chronic renal failure.

The pharmacokinetics of oral diltiazem were studied in 10 patients with chronic renal failure not requiring dialysis and in five healthy volunteers after a single dose of 120 mg. We found that patients with chronic renal failure had lower amounts of unchanged diltiazem and of its main metabolite (MA) in urine and a trend to have slightly higher values of plasma concentration. Since the terminal elimination phase is not affected by chronic renal failure we conclude that this trend is probably the result of alterations in the volume of distribution of diltiazem in these patients.

Placebo-controlled comparative study of the anxiolytic activity and of the pharmacokinetics of oral and sublingual lorazepam in generalized anxiety.

In a randomized crossover study, twelve patients presenting a generalized anxiety disorder received either a 1 mg oral tablet or a 1 mg sublingual tablet of lorazepam three times daily for 7 days. After a 7-day washout period, each patient received a 7-day treatment with the other tablet form. Treatments were administered in a double-blind manner using placebos of both the oral and sublingual tablets. Psychiatric evaluations were carried out before and following each of the three periods. Blood samples were drawn at intervals for 48 h following the last dose of each treatment. The plasma concentrations of lorazepam were measured by gas chromatography using an electron-capture detector. Both the oral and sublingual lorazepam produced a significant anxiolytic effect; there was no statistically significant difference between the therapeutic effectiveness of the two forms of lorazepam. The main pharmacokinetic parameters for the oral and sublingual tablets were, respectively: elimination half-life 15.6h and 11.7h; maximal concentration 40.8 ng ml-1 and 42.2 ng ml-1; time to reach maximal concentration 1.2h and 1.4h; corrected area under the curve 310.6 ng hml-1 and 313.6 ng hml-1. There was no statistically significant difference between the oral and sublingual tablets for any of the pharmacokinetic parameters measured.

Effect of clavulanic acid on susceptibility of Campylobacter jejuni and Campylobacter coli to eight beta-lactam antibiotics.

The effect of clavulanic acid on the susceptibility of 32 strains of Campylobacter jejuni and Campylobacter coli to eight beta-lactam agents was studied. Almost all strains tested became susceptible to amoxicillin and ticarcillin with 1 microgram of clavulanic acid per ml. This compound had little or no effect on susceptibility to penicillin G, cephalothin, cefamandole, and cefoxitin. Clavulanic acid had a marginal effect on cefotaxime and moxalactam susceptibility.

The effect of administration of phenytoin on the pharmacokinetics of isoxicam.

This study was designed to determine whether the disposition of isoxicam is influenced by the coadministration of another acidic drug, highly bound to plasma proteins and extensively metabolized, i.e., phenytoin. Ten healthy volunteers received an oral dose of 200 mg of isoxicam prior to and following the oral administration of phenytoin (100 mg) twice a day for 10 days. Eleven blood samples were drawn during the period following each dose of isoxicam. The area under the isoxicam plasma concentration-time curve (AUC infinity) increased from 389 +/- 66 to 464 +/- 62 micrograms h ml-1 (+/- SEM) (p less than 0.05) after treatment with phenytoin. This increase was due to an increase in isoxicam bioavailability; the absorption rate constant for isoxicam increased correspondingly from 0.34 +/- 0.06 to 1.16 +/- 0.38 h-1 (p less than 0.05). Distribution and clearance of isoxicam were probably not affected as its half-life was not changed, its plasma peak concentration increased, and the time to reach this peak decreased. It is concluded that phenytoin increases the rate and extent of absorption of isoxicam.