Mannose receptor (MR) and Toll-like receptor 2 (TLR2) influence phagosome maturation during Leishmania infection.

Leishmania enter macrophages through receptor-mediated phagocytosis and survive the harsh environment of a phagolysosome. Here, we investigated the interaction between mannose receptor (MR), Toll-like receptor 2 (TLR2), and Leishmania, and the subsequent impact on phagosome maturation. Leishmania parasites are able to delay phagosome maturation, not reaching full maturation until 5 hours post-engulfment. Here, maturation of Leishmania major- and Leishmania donovani-containing phagosomes proceeded as expected in the WT macrophages becoming LAMP1 positive by 6 hours. Interestingly, MR-/- macrophages become LAMP1 positive by ~2 hours and ~4 hours post-infection Leishmania-containing phagosomes lost LAMP1 expression and gained the early marker EEA1. LAMP1 expression was again observed by 6 hours. Leishmania LPG was essential for the delay in both WT and MR-/- macrophages but was not essential for the early maturation (2 hours) observed in MR-/- macrophages. Serum opsonization of Leishmania prior to infection induced identical phagosome maturation patterns in WT and MR-/- macrophages. In the absence of MyD88 or TLR2 on macrophages, Leishmania phagosomes matured significantly faster, becoming LAMP1 positive by ~1-2 hours. These studies add to the knowledge that phagosome maturation is influenced by multiple receptor-ligand interactions and signalling pathways.

Caspase-1 cleavage of transcription factor GATA4 and regulation of cardiac cell fate.

Caspase-1 or interleukin-1ß (IL-1ß) converting enzyme is a pro-inflammatory member of the caspase family. An IL-1ß-independent role for caspase-1 in cardiomyocyte cell death and heart failure has emerged but the mechanisms underlying these effects are incompletely understood. Here, we report that transcription factor GATA4, a key regulator of cardiomyocyte survival and adaptive stress response is an in vivo and in vitro substrate for caspase-1. Caspase-1 mediated cleavage of GATA4 generates a truncated protein that retains the ability to bind DNA but lacks transcriptional activation domains and acts as a dominant negative regulator of GATA4. We show that caspase-1 is rapidly activated in cardiomyocyte nuclei treated with the cell death inducing drug Doxorubicin. We also find that inhibition of caspase-1 alone is as effective as complete caspase inhibition at rescuing GATA4 degradation and myocyte cell death. Caspase-1 inhibition of GATA4 transcriptional activity is rescued by HSP70, which binds directly to GATA4 and masks the caspase recognition motif. The data identify a caspase-1 nuclear substrate and suggest a direct role for caspase-1 in transcriptional regulation. This mechanism may underlie the inflammation-independent action of caspase-1 in other organs.

Leishmania major inhibits IL-12 in macrophages by signalling through CR3 (CD11b/CD18) and down-regulation of ETS-mediated transcription.

Leishmania major is an aetiological agent of cutaneous leishmaniasis. The parasite primarily infects immune sentinel cells, specifically macrophages and dendritic cells, in the mammalian host. Infection is receptor mediated and is known to involve parasite binding to cell surface protein complement receptor 3 (CR3, Mac-1, CD11b/CD18). Engagement of CR3 by various ligands inhibits production of interleukin-12 (IL-12), the cytokine that drives antileishmanial T helper 1-type immune responses. Likewise, L. major infection inhibits IL-12 production and activation of host macrophages. Our data indicate that in the absence of CR3, L. major-infected bone marrow-derived macrophages produce more IL-12 and nitric oxide compared with WT cells upon lipopolysaccharide (LPS) stimulation. We therefore investigated multiple signalling pathways by which L. major may inhibit IL-12 transcription through CR3 ligation. We demonstrate that L. major infection does not elicit significant NFκB p65, MAPK, IRF-1 or IRF-8 activation in WT or CD11b-deficient macrophages. Furthermore, infection neither inhibits LPS-induced MAPK or NFκB activation nor blocks IFN-γ-activated IRF-1 and IRF-8. ETS-mediated transcription, however, is inhibited by L. major infection independently of CR3. Our data indicate that L. major-mediated inhibition of IL-12 occurs through CR3 engagement; however, the mechanism of inhibition is independent of NFκB, MAPK, IRF and ETS.

Natural variation of drug susceptibility in wild-type human immunodeficiency virus type 1.

Wild-type viruses from the ViroLogic phenotype-genotype database were evaluated to determine the upper confidence limit of the drug susceptibility distributions, or "biological cutoffs," for the PhenoSense HIV phenotypic drug susceptibility assay. Definition of the natural variation in drug susceptibility in wild-type human immunodeficiency virus (HIV) type 1 isolates is necessary to determine the prevalence of innate drug resistance and to assess the capability of the PhenoSense assay to reliably measure subtle reductions in drug susceptibility. The biological cutoffs for each drug, defined by the 99th percentile of the fold change in the 50% inhibitory concentration distributions or the mean fold change plus 2 standard deviations, were lower than those previously reported for other phenotypic assays and lower than the clinically relevant cutoffs previously defined for the PhenoSense assay. The 99th percentile fold change values ranged from 1.2 (tenofovir) to 1.8 (zidovudine) for nucleoside reverse transcriptase RT inhibitors (RTIs), from 3.0 (efavirenz) to 6.2 (delavirdine) for nonnucleoside RTIs, and from 1.6 (lopinavir) to 3.6 (nelfinavir) for protease inhibitors. To evaluate the potential role of intrinsic assay variability in the observed variations in the drug susceptibilities of wild-type isolates, 10 reference viruses with different drug susceptibility patterns were tested 8 to 30 times each. The median coefficients of variation in fold change for the reference viruses ranged from 12 to 18% for all drugs except zidovudine (32%), strongly suggesting that the observed differences in wild-type virus susceptibility to the different drugs is related to intrinsic virus variability rather than assay variability. The low biological cutoffs and assay variability suggest that the PhenoSense HIV assay may assist in defining clinically relevant susceptibility cutoffs for resistance to antiretroviral drugs.

Inhibition of purified recombinant reverse transcriptase from wild-type and zidovudine-resistant clinical isolates of human immunodeficiency virus type 1 by zidovudine, stavudine, and lamivudine triphosphates.

Cross-resistance between zidovudine, stavudine, and lamivudine was studied, using purified recombinant reverse transcriptase from a zidovudine-susceptible and -resistant pair of clinical isolates of human immunodeficiency virus type 1. The zidovudine-resistant isolate exhibited low-level cross-resistance to both stavudine and lamivudine in drug susceptibility assays. Enzyme from the resistant isolate demonstrated reduced inhibition by zidovudine triphosphate and stavudine triphosphate and, to a lesser extent, lamivudine triphosphate. These findings provide additional evidence at the viral and enzyme level for cross-resistance between zidovudine and stavudine, and they suggest a possible effect of zidovudine resistance on susceptibility to lamivudine.

Solid-liquid extraction laser excited time-resolved Shpol'skii spectrometry: a facile method for the direct detection of 15 priority pollutants in water samples.

A unique method for monitoring polycyclic aromatic hydrocarbons is reported for routine analysis of water samples. The assay consists of a three-step procedure. One hundred milliliters of water is processed through an octadecyl extraction membrane via solid-liquid extraction. The pollutants are eluted with 5 mL of n-hexane and directly determined in the eluting solvent by laser excited time-resolved Shpol'skii spectrometry. Seventy-seven K fluorescence measurements are made with the aid of an optical fiber probe that avoids the complications of classical low-temperature methodology. The total analysis time from the extraction to PAH identification is approximately 5 min per sample. Limits of detection are at the subparts per billion levels. The simplicity of the experimental procedure, the short analysis time, the selectivity, and the excellent analytical figures of merit demonstrate the advantages of this approach for routine analysis of water samples.

Phenotypic hypersusceptibility to non-nucleoside reverse transcriptase inhibitors in treatment-experienced HIV-infected patients: impact on virological response to efavirenz-based therapy.

BACKGROUND: Enhanced susceptibility to non-nucleoside reverse transcriptase inhibitors (NNRTI) was recently described in association with increased resistance to nucleoside analogs (nucleoside reverse transcriptase inhibitors; NRTI). OBJECTIVES: To determine the prevalence of NNRTI hypersusceptibility, the genotypic correlates, and its impact on virologic response to efavirenz-based salvage therapy. METHODS: Genotype and phenotype testing was performed retrospectively on baseline isolates from 30 patients who received salvage therapy containing efavirenz. NNRTI hypersusceptibility was defined as a 50% inhibitory concentration (IC(50)) of < 0.5 that of the wild-type control. RESULTS: Eight isolates had major NNRTI mutations. Among the 22 isolates with no major NNRTI mutations, 11 (50%) were hypersusceptible to efavirenz, 10 (45%) to delavirdine, and eight (36%) to nevirapine. Among eight isolates with NNRTI mutations, NNRTI resistance was present, but at lower than expected levels. The number of NRTI mutations was correlated inversely with the fold decrease in susceptibility to efavirenz (Spearman's rho, -0.57; P = 0.005), delavirdine (rho, -0.43; P = 0.04), and nevirapine (rho, -0.69; P < 0.001). Excluding subjects with NNRTI mutations, subjects with efavirenz hypersusceptibility at baseline had significantly better virologic suppression over 24 weeks than those without efavirenz hypersusceptibility (P < 0.001). CONCLUSION: NNRTI hypersusceptibility is common in heavily treated but NNRTI naive patients and is related directly to NRTI resistance mutations. Among patients receiving efavirenz-containing regimens, NNRTI hypersusceptibility was associated with an improved virologic outcome after 24 weeks of therapy. A reversal of phenotypic resistance was seen in patients with NNRTI mutations in the presence of multiple NRTI mutations, but no obvious virologic benefit of this phenomenon was seen in this study.

Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA.

We have determined the 3.0 A resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The 'RNase H primer grip', consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual 'unzipping' of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.

Treating patients like customers. Just-in-time inventory control for patient-centered care.

Managing change in health care is a complex, poorly studied process that's even more poorly understood. We do not have a clear model to visualize as we contemplate just what it is we are changing. Explore how modern hospitals are "warehousing" patients like excess inventory, and examine the changes needed to escape this morass. A strong physician/hospital alliance is the key to establishing more efficient, patient-centered care.

Screening potential of solid-phase extraction and solid surface room temperature fluorimetry for polycyclic aromatic hydrocarbons in water samples.

The analytical potential of solid-phase extraction and room temperature fluorimetry for screening polycylic aromatic hydrocarbons in water samples is evaluated. Solid-phase extraction was performed via a syringe procedure previously reported (Talanta 52 (2000) 727). The simplicity of fluorescence measurements on the solid substrate is equivalent to solution measurements. Since oxygen quenching of fluorescence is not significant, placing the extraction membrane in the substrate holder of the spectrometer rapidly performs fluorescence measurements. Limits of detection at the pg ml(-1) level were estimated for several pollutants. With a commercial spectrofluorimeter, benzo(a)pyrene was quantitatively determined at the 5 pg ml(-1) concentration level. This concentration compared favorably to limits of detection estimated by laser-induced fluorimetry. A unique advantage of this approach was the possibility of adjusting the volume of extracted water to reach concentration levels below instrumental detection levels. Since SPE procedure was rapid and simple the trade-off of including an additional experimental step to lower limits of detection was advantageous. Although the selectivity of this approach was not fully investigated, our studies showed that selective excitation was sufficient to identify benzo(a)pyrene in a seven-component mixture and spiked Red River water of unknown composition.

Randomized trial of oral hydrocortisone and its effect on emergency physicians during night duty.

STUDY OBJECTIVE: To analyze the effects of hydrocortisone (40 mg. p.o.) administered to emergency physicians on their first night shift following a series of day shifts. DESIGN: Prospective, double-blinded internal crossover study on objective and subjective parameters. Each participant was studied for a minimum of 10 nights. TYPE OF PARTICIPANTS: Four healthy male emergency physicians in their mid to late thirties. INTERVENTIONS: After baseline endocrine assessment, the subjects ingested a capsule containing either 40 mg of hydrocortisone or placebo (lactose) at the start of a first nightshift (starting at 10 pm or 11 pm) after day duty. Subjects self-administered psychological testing one hour after taking an oral capsule by listening to a self-guided audio tape (between 11 and 12 p.m), and again between 4 and 5 am. Blood samples were obtained during the first 4 nights of each subject at 11 pm, 2, 5 and 8 am. MEASUREMENTS AND MAIN RESULTS: Four emergency physicians entered 42 nights of data. No differences in testing were detected. Plasma cortisol levels were measured and demonstrated cortisol levels consistent with oral replacement therapy. Physicians could subjectively differentiate the difference between hydrocortisone treatment and placebo: of 21 hydrocortisone nights, 17 were identified as "a good night" in reference to fatigue. Of 21 nights without hydrocortisone, 15 were identified as "bad" nights, (p < .001). CONCLUSION: Hydrocortisone, administered before a nightshift to day-accommodated workers, recreated the rise of plasma cortisol seen on awakening and was shown to be an effective means of decreasing subjective fatigue of a first nightshift.

Reduced susceptibility of human immunodeficiency virus type 1 (HIV-1) from patients with primary HIV infection to nonnucleoside reverse transcriptase inhibitors is associated with variation at novel amino acid sites.

Recently, significant numbers of individuals with primary human immunodeficiency virus (HIV) infection have been found to harbor viral strains with reduced susceptibility to antiretroviral drugs. In one study, HIV from 16% of such antiretroviral-naive individuals was shown to have a susceptibility to nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) between 2.5- and 10-fold lower than that of a wild-type control. Mutations in the RT domain that had previously been associated with antiretroviral resistance were not shared by these strains. We have analyzed by logistic regression 46 variable amino acid sites in RT for their effect on susceptibility and have identified two novel sites influencing susceptibility to NNRTIs: amino acids 135 and 283 in RT. Eight different combinations of amino acids at these sites were observed among these patients. These combinations showed a 14-fold range in mean susceptibility to both nevirapine and delavirdine. In vitro mutagenesis of the control strain combined with a phenotypic assay confirmed the significance of amino acid variation at these sites for susceptibility to NNRTIs.

A new pathway to product standardization.

As the benefits of product standardization become more evident in improved financial, managerial, and clinical outcomes, tools to make the process easier will be in demand. Once a standardization program is established, e-commerce offers tools to keep it on track.

A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1.

Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. We have developed a novel phenotypic drug susceptibility assay that may be useful in guiding therapy and improving long-term suppression of HIV replication. Susceptibility to protease (PR) and reverse transcriptase (RT) inhibitors is measured by using resistance test vectors (RTVs) that contain a luciferase indicator gene and PR and RT sequences derived from HIV-1 in patient plasma. Cells are transfected with RTV DNA, resulting in the production of virus particles that are used to infect target cells. Since RTVs are replication defective, luciferase activity is measured following a single round of replication. The assay has been automated to increase throughput and is completed in 8 to 10 days. Test results may be useful in facilitating the selection of optimal treatment regimens for patients who have failed prior therapy or drug-naive patients infected with drug-resistant virus. In addition, the assay can be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant strains of HIV-1.

Drug susceptibility in HIV infection after viral rebound in patients receiving indinavir-containing regimens.

CONTEXT: Loss of viral suppression in patients infected with human immunodeficiency virus (HIV), who are receiving potent antiretroviral therapy, has been attributed to outgrowth of drug-resistant virus; however, resistance patterns are not well characterized in patients whose protease inhibitor combination therapy fails afterachieving viral suppression. OBJECTIVE: To characterize drug susceptibility of virus from HIV-infected patients who are failing to sustain suppression while taking an indinavir-containing antiretroviral regimen. DESIGN AND SETTING: Substudy of the AIDS Clinical Trials Group 343, a multicenter clinical research trial conducted between February 1997 and October 1998. PATIENTS: Twenty-six subjects who experienced rebound (HIV RNA level > or =200 copies/mL) during indinavir monotherapy (n = 9) or triple-drug therapy (indinavir, lamivudine, and zidovudine; n = 17) after initially achieving suppression while receiving all 3 drugs, and 10 control subjects who had viral suppression while receiving triple-drug therapy. MAIN OUTCOME MEASURE: Drug susceptibility, determined by a phenotypic assay and genotypic evidence of resistance assessed by nucleotide sequencing of protease and reverse transcriptase, compared among the 3 patient groups. RESULTS: Indinavir resistance was not detected in the 9 subjects with viral rebound during indinavir monotherapy or in the 17 subjects with rebound during triple-drug therapy, despite plasma HIV RNA levels ranging from 10(2) to 10(5) copies/mL. In contrast, lamivudine resistance was detected by phenotypic assay in rebound isolates from 14 of 17 subjects receiving triple-drug therapy, and genotypic analyses showed changes at codon 184 of reverse transcriptase in these 14 isolates. Mean random plasma indinavir concentrations in the 2 groups with rebound were similar to those of a control group with sustained viral suppression, although levels below 50 ng/mL were more frequent in the triple-drug group than in the control group (P = .03). CONCLUSIONS: Loss of viral suppression may be due to suboptimal antiviral potency, and selection of a predominantly indinavir-resistant virus population may be delayed for months even in the presence of ongoing indinavir therapy. The results suggest possible value in assessing strategies using drug components of failing regimens evaluated with resistance testing.

Reduced antiretroviral drug susceptibility among patients with primary HIV infection.

CONTEXT: The transmission of drug-resistant human immunodeficiency virus (HIV) has been documented, but the prevalence of such transmission is unknown. OBJECTIVE: To assess the spectrum and frequency of antiretroviral susceptibility among subjects with primary HIV infection. DESIGN, SETTING, AND PATIENTS: Retrospective analysis of 141 subjects identified from clinical research centers in 5 major metropolitan areas, enrolled from 1989 to 1998, with HIV seroconversion within the preceding 12 months and no more than 7 days' prior antiretroviral (ARV) therapy. MAIN OUTCOME MEASURES: Phenotypic and genotypic ARV susceptibility of HIV from plasma samples. RESULTS: The transmission of drug-resistant HIV as assessed by a greater than 10-fold reduction in ARV susceptibility to 1 or more drugs was observed in 3 (2%) of 141 subjects, including to a nonnucleoside reverse transcriptase inhibitor in 1 patient and to a nucleoside reverse transcriptase inhibitor and a protease inhibitor in 2 patients. Population-based sequence analysis of these 3 samples identified multidrug-resistance mutations in reverse transcriptase (M184V, T215Y, K219K/R) and protease (L101/V, K20R, M361, M46I, G48V, L63P, A71T, V771, V82T, 184V, L90M) in the 2 latter patient samples, along with numerous polymorphisms. A reduction in susceptibility of greater than 2.5- to 10-fold to 1 or more drugs was observed in viral isolates from 36 patients (26%). Sequence analysis of these 36 samples identified well-characterized drug resistance mutation in reverse transcriptase and protease in only 1 of these patients. CONCLUSIONS: Reductions in drug susceptibility of more than 10-fold were rare among this cohort of recently HIV-infected subjects and were distributed among each of the 3 major classes of ARV drugs tested. Reductions in susceptibility of more than 2.5- to 10-fold to certain ARV drugs of unknown clinical significance were highly prevalent among newly infected patients. Resistance testing may be warranted to monitor the frequency of drug resistance over time and to assess the potential for geographic variability.

The EV-O-derived cell line DF-1 supports the efficient replication of avian leukosis-sarcoma viruses and vectors.

The lack of a well-behaved permanent, adherent, nontransformed chicken cell line has made some experiments with avian leukosis-sarcoma viruses (ASLV) and vectors considerably more difficult. The EV-O-derived line, DF-1, supports the efficient replication of subgroups (A), (B), and (C) ASLV, as well as amphotrophic murine leukemia virus and an ASLV-derived vector that has its env gene derived from the env gene from an amphotrophic murine leukemia virus. The cell line responds appropriately to the expression of a transforming oncogene (v-myc) to a growth suppressor gene [p21(waf1)] and can be sorted (using FACS) if infected by an ASLV vector that expresses GFP.

Production of avian leukosis virus particles in mammalian cells can be mediated by the interaction of the human immunodeficiency virus protein Rev and the Rev-responsive element.

In human immunodeficiency virus type 1-infected cells, the efficient expression of viral proteins from unspliced and singly spliced RNAs is dependent on two factors: the presence in the cell of the viral protein Rev and the presence in the viral RNA of the Rev-responsive element (RRE). We show here that the HIV-1 Rev/RRE system can increase the expression of avian leukosis virus (ALV) structural proteins in mammalian cells (D-17 canine osteosarcoma) and promote the release of mature ALV virions from these cells. In this system, the Rev/RRE interaction appears to facilitate the export of full-length unspliced ALV RNA from the nucleus to the cytoplasm, allowing increased production of the ALV structural proteins. Gag protein is produced in the cytoplasm of the ALV-transfected cells even in the absence of a Rev/RRE interaction. However, a functional Rev/RRE interaction increases the amount of Gag present intracellularly and, more strikingly, results in the release of mature ALV particles into the supernatant. RCAS virus containing an RRE is replication-competent in chicken embryo fibroblasts; however, we have been unable to determine whether the particles produced in D-17 cells are as infectious as the particles produced in chicken embryo fibroblasts.

Replication of avian leukosis viruses with mutations at the primer binding site: use of alternative tRNAs as primers.

We have tested whether avian leukosis viruses (ALVs) can use tRNAs other than tRNATrp to initiate reverse transcription. The primer binding site (PBS) of a wild-type ALV provirus, which is complementary to the 3' end of tRNA(Trp), was replaced with sequences homologous to the 3' ends of six different chicken tRNAs (tRN(APro), tRNA(Lys), tRNA(Met), tRNA(Ile), tRNA(Phe), and tRNA(Ser)). Transfection of these proviruses into chicken embryo fibroblasts resulted in the production of infectious viruses, all of which apparently used the tRNA specified by the mutated PBS to replicate. However, growth of these viruses resulted in reversion to the wild-type (tRNA(Trp)) PBS. Some of the viruses revert quite quickly, while others are more stable. The relative stability of a given PBS correlated with the concentration of the corresponding tRNA in the virion. We determined the percentage of viral RNA that had a tRNA bound to the PBS and found that the occupancy rate is lower in the mutants than in the wild-type virus. We conclude that many different tRNAs can be used as primers to initiate reverse transcription in ALV. However, ALVs that use tRNA(Trp) have a growth advantage over ALVs that use other tRNAs.