Mycobacterium tuberculosis sterilizing activity of faropenem, pyrazinamide and linezolid combination and failure to shorten the therapy duration.

BACKGROUND: Faropenem (F), an orally bioavailable ß-lactam, kills Mycobacterium tuberculosis (Mtb) without the help of a ß-lactamase inhibitor. This study explored the sterilizing effect of adding F once or twice daily to a linezolid (L) plus pyrazinamide (Z) backbone regimen. METHODS: In vitro studies were performed using the hollow fiber model of tuberculosis (HFS-TB) to compare the kill rates of: 1) ZL two-drug combination; 2) F administered once daily plus ZL (F1ZL); 3) F administered twice-daily plus once daily ZL (F2ZL); 4) F2ZL with high-dose Z (F2ZhiL); 5) standard therapy of isoniazid, rifampin and Z; and 6) non-treated controls. The study was performed over 56 days with three HFS-TB replicates for each regimen. RESULTS: Mtb in the non-treated HFS-TB grew at a rate of 0.018 ± 0.007 log10 CFU/mL/day. The exponential kill rates for standard therapy were 6.6-13.2-fold higher than ZL dual therapy. The F1ZL and F2ZL regimens ranked third. The pre-existing isoniazid-resistant sub-population in the inoculum (1.34 ± 0.57 log10 CFU/mL) grew to 4.21 ± 0.58 log10 CFU/mL in 56 days in non-treated HFS-TB. However, no isoniazid-resistant sub-population was recorded in any of the FZL combination regimens. CONCLUSION: Due to the slow kill rate compared to standard therapy, FZL regimens are unlikely to shorten therapy duration. Efficacy of these regimens against drug-resistant tuberculosis needs to be determined.

A 'shock and awe' thioridazine and moxifloxacin combination-based regimen for pulmonary Mycobacterium avium-intracellulare complex disease.

OBJECTIVES: To develop a thioridazine/moxifloxacin-based combination regimen for treatment of pulmonary infection due to Mycobacterium avium-intracellulare complex (MAC) that kills bacteria faster than the standard treatment regimen. METHODS: Monocytes were infected with MAC and inoculated into the hollow-fibre system model for pulmonary MAC disease (HFS-MAC). We co-administered ethambutol plus azithromycin daily for 28 days, to achieve the same human concentration-time profiles that result from standard doses, in three HFS-MAC systems. Two experimental regimens consisted of thioridazine at an exposure associated with optimal kill, given intermittently on days 0, 3, 7 and 10. Regimen A consisted of thioridazine in combination with standard dose azithromycin for the entire study duration. Regimen B was thioridazine plus moxifloxacin at concentration-time profiles achieved by the standard daily dose administered for 14 days, followed by daily azithromycin. Each HFS-MAC was sampled for bacterial burden every 7 days. RESULTS: The bacteria in the non-treated HFS-MAC grew at a rate of 0.11 ±âŸ0.01 log10 cfu/mL/day. The azithromycin/ethambutol regimen decreased bacterial burden by 1.21 ±âŸ0.74 log10 cfu/mL below baseline during the first 7 days, after which it failed. Regimen A killed 3.28 ±âŸ0.32 log10 cfu/mL below baseline up to day 14, after which regrowth occurred once thioridazine treatment stopped. Regimen B killed bacteria to below the limits of detection in 7 days (≥5.0 log10 cfu/mL kill), with rebound in the azithromycin continuation phase. CONCLUSIONS: The thioridazine/moxifloxacin regimen demonstrated that rapid microbial kill could be achieved within 7 days. This is a proof of principle that short-course chemotherapy for pulmonary MAC is possible.

A Long-term Co-perfused Disseminated Tuberculosis-3D Liver Hollow Fiber Model for Both Drug Efficacy and Hepatotoxicity in Babies.

Treatment of disseminated tuberculosis in children≤6years has not been optimized. The pyrazinamide-containing combination regimen used to treat disseminated tuberculosis in babies and toddlers was extrapolated from adult pulmonary tuberculosis. Due to hepatotoxicity worries, there are no dose-response studies in children. We designed a hollow fiber system model of disseminated intracellular tuberculosis with co-perfused three-dimensional organotypic liver modules to simultaneously test for efficacy and toxicity. We utilized pediatric pharmacokinetics of pyrazinamide and acetaminophen to determine dose-dependent pyrazinamide efficacy and hepatotoxicity. Acetaminophen concentrations that cause hepatotoxicity in children led to elevated liver function tests, while 100mg/kg pyrazinamide did not. Surprisingly, pyrazinamide did not kill intracellular Mycobacterium tuberculosis up to fourfold the standard dose as monotherapy or as combination therapy, despite achieving high intracellular concentrations. Host-pathogen RNA-sequencing revealed lack of a pyrazinamide exposure transcript signature in intracellular bacteria or of phagolysosome acidification on pH imaging. Artificial intelligence algorithms confirmed that pyrazinamide was not predictive of good clinical outcomes in children≤6years who had extrapulmonary tuberculosis. Thus, adding a drug that works inside macrophages could benefit children with disseminated tuberculosis. Our in vitro model can be used to identify such new regimens that could accelerate cure while minimizing toxicity.

Amikacin Pharmacokinetics/Pharmacodynamics in a Novel Hollow-Fiber Mycobacterium abscessus Disease Model.

The treatment of pulmonary Mycobacterium abscessus disease is associated with very high failure rates and easily acquired drug resistance. Amikacin is the key drug in treatment regimens, but the optimal doses are unknown. No good preclinical model exists to perform formal pharmacokinetics/pharmacodynamics experiments to determine these optimal doses. We developed a hollow-fiber system model of M. abscessus disease and studied amikacin exposure effects and dose scheduling. We mimicked amikacin human pulmonary pharmacokinetics. Both amikacin microbial kill and acquired drug resistance were linked to the peak concentration-to-MIC ratios; the peak/MIC ratio associated with 80% of maximal kill (EC80) was 3.20. However, on the day of the most extensive microbial kill, the bacillary burden did not fall below the starting inoculum. We performed Monte Carlo simulations of 10,000 patients with pulmonary M. abscessus infection and examined the probability that patients treated with one of 6 doses from 750 mg to 4,000 mg would achieve or exceed the EC80. We also examined these doses for the ability to achieve a cumulative area under the concentration-time curve of 82,232 mg · h/liter × days, which is associated with ototoxicity. The standard amikacin doses of 750 to 1,500 mg a day achieved the EC80 in ≤ 21% of the patients, while a dose of 4 g/day achieved this in 70% of the patients but at the cost of high rates of ototoxicity within a month or two. The susceptibility breakpoint was an MIC of 8 to 16 mg/liter. Thus, amikacin, as currently dosed, has limited efficacy against M. abscessus. It is urgent that different antibiotics be tested using our preclinical model and new regimens developed.

Rapid drug tolerance and dramatic sterilizing effect of moxifloxacin monotherapy in a novel hollow-fiber model of intracellular Mycobacterium kansasii disease.

Mycobacterium kansasii is the second most common mycobacterial cause of lung disease. Standard treatment consists of rifampin, isoniazid, and ethambutol for at least 12 months after negative sputum. Thus, shorter-duration therapies are needed. Moxifloxacin has good MICs for M. kansasii. However, good preclinical models to identify optimal doses currently are lacking. We developed a novel hollow fiber system model of intracellular M. kansasii infection. We indexed the efficacy of the standard combination regimen, which was a kill rate of -0.08 ± 0.05 log10 CFU/ml/day (r(2) = 0.99). We next performed moxifloxacin dose-effect and dose-scheduling studies at a half-life of 11.1 ± 6.47 h. Some systems also were treated with the efflux pump inhibitor reserpine. The highest moxifloxacin exposure, as well as lower exposures plus reserpine, sterilized the cultures by day 7. This suggests that efflux pump-mediated tolerance at low ratios of the area under the concentration-time curve from 0 to 24 h (AUC0 - 24) to MICs is an early bacterial defense mechanism but is overcome by higher exposures. The highest rate of moxifloxacin monotherapy sterilization was -0.82 ± 0.15 log10 CFU/ml/day (r(2) = 0.97). The moxifloxacin exposure associated with 80% of maximal kill (EC80) was an AUC0-24/MIC of 317 (the non-protein-bound moxifloxacin AUC0-24/MIC was 158.5). We performed Monte Carlo simulations of 10,000 patients in order to identify the moxifloxacin dose that would achieve or exceed the EC80. The simulations revealed an optimal moxifloxacin dose of 800 mg a day. The MIC susceptibility breakpoint at this dose was 0.25 mg/liter. Thus, moxifloxacin, at high enough doses, is suitable to study in patients for the potential to add rapid sterilization to the standard regimen.

Sperm morphology in the Malagasy rodents (Muroidea: Nesomyinae).

The morphology of the spermatozoon of representative species of the subfamily Nesomyinae (Muroidea: Nesomyidae), a monophyletic group of rodents endemic to Madagascar, was examined by light and electron microscopy to determine the sperm head shape and tail length across the species. Marked interspecific differences were found to occur in both the form of the sperm head and length of the tail. The species that possess a sperm head with an apical hook, which largely contains acrosomal material, generally displayed longer sperm tails, and a species with a spatulate sperm head had the shortest tail. The association between sperm head shape and tail length mirrors that previously found in Eurasian and Australasian murine rodents. Thus, the repeated association between sperm head shape and tail length across these groups of muroid rodents clearly indicates a functional relationship between these two features. A comparison of sperm morphology of the nesomyines to that of related muroid rodents on the mainland of Africa suggests that the possession of an apical hook is the ancestral condition.

Branching out to gain control: how the pre-TCR is linked to multiple functions.

How is signaling specificity achieved by the pre-TCR during selection of T-cell fate? Like the TCR, this receptor controls many functions, and recent studies define which pathways couple the pre-TCR to the molecular events controlling survival, proliferation, allelic exclusion at the TCRbeta locus, and further differentiation.

Identification of a novel pre-TCR isoform in which the accessibility of the TCR beta subunit is determined by occupancy of the 'missing' V domain of pre-T alpha.

We have identified a novel pre-TCR isoform that is structurally distinct from conventional pre-TCR complexes and whose TCR beta chains are inaccessible to anti-TCR beta antibodies. We term this pre-TCR isoform the MB (masked beta)-pre-TCR. Pre-T alpha (pT alpha) subunits of MB-pre-TCR complexes have a larger apparent mol. wt due to extensive modification with O:-linked carbohydrates; however, preventing addition of O-glycans does not restore antibody recognition of the TCR beta subunits of MB-pre-TCR complexes. Importantly, accessibility of TCR beta chains in MB-pre-TCR complexes is restored by filling in the 'missing' variable (V) domain of pT alpha with a V domain from TCR alpha. Moreover, the proportion of pre-TCR complexes in which the TCR beta subunits are accessible to anti-TCR beta antibody varies with the cellular context, suggesting that TCR beta accessibility is controlled by a trans-acting factor. The way in which this factor might control TCR beta accessibility as well as the physiologic relevance of TCR beta masking for pre-TCR function are discussed.

Control of early thymocyte development by the pre-T cell receptor complex: A receptor without a ligand?

Beta-selection refers to a developmental checkpoint linking thymocyte survival to the outcome of antigen receptor gene rearrangement. Immature thymocytes that productively rear-range the gene segments of the TCRbeta locus undergo proliferative expansion and mature to the CD4(+)CD8(+)stage; those failing to do so die by apoptosis. How are these precursor cells alerted that TCRbeta rearrangement has been productive? While it is clear that this process involves signals transduced by a surrogate form of the TCR termed the pre-TCR, it remains unclear how pre-TCR signals are triggered. In this review, we will discuss the implications of recent experimental attempts to address this issue, as well as how pre-TCR activation is linked to the changes in gene expression that underlie thymocyte development.

Signals transduced by CD3epsilon, but not by surface pre-TCR complexes, are able to induce maturation of an early thymic lymphoma in vitro.

Development of immature CD4-CD8- (double-negative) thymocytes to the CD4+CD8+ (double-positive) stage is linked to productive rearrangement of the TCRbeta locus by signals transduced through the pre-TCR. However, the mechanism whereby pre-TCR signaling is initiated remains unclear, in part due to the lack of an in vitro model system amenable to both biochemical and genetic analysis. In this study, we establish the thymic lymphoma Scid.adh as such a model system. Scid.adh responds to Ab engagement of surface IL-2Ra (TAC):CD3epsilon molecules (a signaling chimera that mimics pre-TCR signaling in vivo) by undergoing changes in gene expression observed following pre-TCR activation in normal thymocytes. These changes include down-regulation of CD25, recombinase-activating gene (RAG)-1, RAG-2, and pTalpha; and the up-regulation of TCRalpha germline transcripts. We term this complete set of changes in gene expression, in vitro maturation. Interestingly, Scid.adh undergoes only a subset of these changes in gene expression following Ab engagement of the pre-TCR. Our findings make two important points. First, because TAC:CD3epsilon stimulation of Scid.adh induces physiologically relevant changes in gene expression, Scid.adh is an excellent cellular system for investigating the molecular requirements for pre-TCR signaling. Second, Ab engagement of CD3epsilon signaling domains in isolation (TAC:CD3epsilon) promotes in vitro maturation of Scid.adh, whereas engagement of CD3epsilon molecules contained within the complete pre-TCR fails to do so. Our current working hypothesis is that CD3epsilon fails to promote in vitro maturation when in the context of an Ab-engaged pre-TCR because another pre-TCR subunit(s), possibly TCRzeta, qualitatively alters the CD3epsilon signal.

Stenting with a true bifurcated stent: acute and mid-term follow-up results.

Percutaneous therapy of coronary bifurcated lesions is associated with greater risk of acute complications and late restenosis. Numerous innovative techniques using various stent types have been proposed. We report the first clinical use of a truly bifurcated stent (Bard XT Carina). The implantation procedure, favorable medium-term angiographic and 1-year clinical follow-up of the first human use in a 43-year-old female are illustrated with angiography and intravascular ultrasound. As a single prosthesis that effectively covers the bifurcation, this stent presents appealing alternative for the treatment of coronary bifurcation lesions when compared to methods that involve multiple-stent implantation. Cathet. Cardiovasc. Intervent. 47:361-369, 1999.

The formation of intramolecular disulfide bridges is required for induction of the Sindbis virus mutant ts23 phenotype.

The Sindbis virus envelope protein spike is a hetero-oligomeric complex composed of a trimer of glycoprotein E1-E2 heterodimers. Spike assembly is a multistep process which occurs in the endoplasmic reticulum (ER) and is required for the export of E1 from the ER. PE2 (precursor to E2), however, can transit through the secretory pathway and be expressed at the cell surface in the absence of E1. Although oligomer formation does not appear to be required for the export of PE2, there is evidence that defects in E1 folding can affect PE2 transit from the ER. Temperature-sensitive mutant ts23 of Sindbis virus contains two amino acid substitutions in E1, while PE2 and capsid protein have the wild-type sequence; however, at the nonpermissive temperature, both E1 and PE2 are retained within the ER and can be isolated in protein aggregates with the molecular chaperone GRP78-BiP. We previously demonstrated that the temperature sensitivity for ts23 was lost as oligomer formation took place at the permissive temperature, suggesting that temperature sensitivity is initiated early in the process of viral spike assembly (M. Carleton and D. T. Brown, J. Virol. 70:952-959, 1996). Experiments described herein investigated the defects in envelope protein maturation that occur in ts23-infected cells and which result in retention of both envelope proteins in the ER. The data demonstrate that in ts23-infected cells incubated at the nonpermissive temperature, E1 folding is disrupted early after synthesis, resulting in the rapid incorporation of both E1 and PE2 into disulfide-stabilized aggregates. Furthermore, the aberrant E1 conformation which is responsible for induction of the ts phenotype requires the formation of intramolecular disulfide bridges formed prior to E1 association with PE2 and the completion of E1 folding.

Role of glycoprotein PE2 in formation and maturation of the Sindbis virus spike.

Sindbis virus envelope assembly is a multistep process resulting in the maturation of a rigid, highly ordered T=4 icosahedral protein lattice containing 80 spikes composed of trimers of E1-E2 heterodimers. Intramolecular disulfide bonds within E1 stabilize E1-E1 associations required for envelope formation and maintenance of the envelope's structural integrity. The structural integrity of the envelope protein lattice is resistant to reduction by dithiothreitol (DTT), indicating that E1 disulfides which stabilize structural domains become inaccessible to DTT at some point during virus maturation. The development of E1 resistance to DTT occurs prior to the completion of E1 folding and is temporally correlated with spike assembly in the endoplasmic reticulum. From these data we have predicted that in the final stages of spike assembly, E1 intramolecular disulfides, which stabilize the structural integrity of the envelope protein lattice, are buried within the spike and become inaccessible to the reductive activity of DTT. The spike is formed prior to the completion of E1 folding, and we have suggested that PE2 (the precursor to E2) may play a critical role in E1 folding after PE2-E1 oligomer formation has occurred. In this study we have investigated the role of PE2 in E1 folding, oligomer formation, and development of E1 resistance to both protease digestion and reduction by DTT by using a Sindbis virus replicon (SINrep/E1) which allows for the expression of E1 in the presence of truncated PE2. Through pulse-chase analysis of both Sindbis virus- and SINrep/E1-infected cells, we have determined that the folding of E1 into a trypsin-resistant conformation and into its most compact and stable form is not dependent upon association of E1 with PE2. However, E1 association with PE2 is required for oligomer formation, the export of E1 from the endoplasmic reticulum, and E1 acquisition of resistance to DTT.

Disulfide bridge-mediated folding of Sindbis virus glycoproteins.

The Sindbis virus envelope is composed of 80 E1-E2 (envelope glycoprotein) heterotrimers organized into an icosahedral protein lattice with T=4 symmetry. The structural integrity of the envelope protein lattice is maintained by E1-E1 interactions which are stabilized by intramolecular disulfide bonds. Structural domains of the envelope proteins sustain the envelope's icosahedral lattice, while functional domains are responsible for virus attachment and membrane fusion. We have previously shown that within the mature Sindbis virus particle, the structural domains of the envelope proteins are significantly more resistant to the membrane-permeative, sulfhydryl-reducing agent dithiothreitol (DTT) than are the functional domains (R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330-336, 1992). We have used DTT to probe the accessibility of intramolecular disulfides within PE2 (the precursor to E2) and E1, as these proteins fold and are assembled into the spike heterotrimer. We have determined through pulse-chase analysis that intramolecular disulfide bonds within PE2 are always sensitive to DTT when the glycoproteins are in the endoplasmic reticulum. The reduction of these disulfides results in the disruption of PE2-E1 associations. E1 acquires increased resistance to DTT as it folds through a series of disulfide intermediates (E1alpha, -beta, and -gamma) prior to assuming its native and most compact conformation (E1epsilon). The transition from a DTT-sensitive form into a form which exhibits increased resistance to DTT occurs after E1 has folded into its E1beta conformation and correlates temporally with the dissociation of BiP-E1 complexes and the formation of PE2-E1 heterotrimers. We propose that the disulfide bonds within E1 which stabilize the protein domains required for maintaining the structural integrity of the envelope protein lattice form early within the folding pathway of E1 and become inaccessible to DTT once the heterotrimer has formed.

Events in the endoplasmic reticulum abrogate the temperature sensitivity of Sindbis virus mutant ts23.

Temperature-sensitive mutations in proteins produced at or heated to a nonpermissive temperature render the proteins defective in some aspect of their maturation into functional entities. The characterization of temperature-sensitive mutations in model proteins, such as virus membrane proteins, has allowed the elucidation of critical events in the maturation of virus membranes as well as in the intracellular folding, processing, and transport of membrane proteins in general. We have used a transport-defective, temperature-sensitive mutant of Sindbis virus, ts23, which has two amino acid changes in the envelope protein E1, to further examine requirements placed upon the glycoproteins for their export to the plasma membrane. Pulse-chase experiments in which we utilized the transport inhibitors monensin and brefeldin A allowed us to synthesize and assemble the glycoproteins of ts23 into export-competent heterodimers at the permissive temperature while concurrently blocking their export to the cell surface. After removal of the inhibitors and a shift to the nonpermissive temperature, we assayed for protein transport, cell-cell fusion, and infectious-particle production. Taken together, the data show that the irreversible loss of the temperature-sensitive phenotype of ts23 can be correlated with the folding of E1 and the formation of export-competent PE2-E1 heterodimers in the endoplasmic reticulum. Furthermore, we have found that E1 pairs with PE2 to form the heterodimer prior to the completion of E1 folding.

The vertical file enters the electronic age.

Vertical files are revered institutions in many libraries. The reference staff at the Spencer S. Eccles Health Sciences Library at the University of Utah automated their vertical file and made the information in it accessible via the Library's Integrated Library System. Medical Subject Headings (MeSH) were applied to vertical files and simple records leading to them were entered in the Library's online catalog. Electronic access to the vertical file increases the availability of concepts too new to be in medical books and permits the Library to meet the needs of lay patrons searching for basic information on popular health care topics.