Cumulative Fraction of Response for Once- and Twice-Daily Delamanid in Patients with Pulmonary Multidrug-Resistant Tuberculosis.

Pharmacokinetic (PK) and pharmacodynamic (PD) analyses were conducted to determine the cumulative fraction of response (CFR) for 100 mg twice-daily (BID) and 200 mg once-daily (QD) delamanid in patients with multidrug-resistant tuberculosis (MDR-TB), using a pharmacodynamic target (PDT) that achieves 80% of maximum efficacy. First, in the mouse model of chronic TB, the PK/PD index for delamanid efficacy was determined to be area under the drug concentration-time curve over 24 h divided by MIC (AUC0-24/MIC), with a PDT of 252. Second, in the hollow-fiber system model of tuberculosis, plasma-equivalent PDTs were identified as an AUC0-24/MIC of 195 in log-phase bacteria and 201 in pH 5.8 cultures. Third, delamanid plasma AUC0-24/MIC and sputum bacterial decline data from two early bactericidal activity trials identified a clinical PDT of AUC0-24/MIC of 171. Finally, the CFRs for the currently approved 100-mg BID dose were determined to be above 95% in two MDR-TB clinical trials. The CFR for the 200-mg QD dose, evaluated in a trial in which delamanid was administered as 100 mg BID for 8 weeks plus 200 mg QD for 18 weeks, was 89.3% based on the mouse PDT and >90% on the other PDTs. QTcF (QTc interval corrected for heart rate by Fridericia's formula) prolongation was approximately 50% lower for the 200 mg QD dose than the 100 mg BID dose. In conclusion, while CFRs of 100 mg BID and 200 mg QD delamanid were close to or above 90% in patients with MDR-TB, more-convenient once-daily dosing of delamanid is feasible and likely to have less effect on QTcF prolongation.

d-Cycloserine Pharmacokinetics/Pharmacodynamics, Susceptibility, and Dosing Implications in Multidrug-resistant Tuberculosis: A Faustian Deal.

Background: d-cycloserine is used to treat multidrug-resistant tuberculosis. Its efficacy, contribution in combination therapy, and best clinical dose are unclear, also data on the d-cycloserine minimum inhibitory concentration (MIC) distributions is scant. Methods: We performed a systematic search to identify pharmacokinetic and pharmacodynamic studies performed with d-cycloserine. We then performed a combined exposure-effect and dose fractionation study of d-cycloserine in the hollow fiber system model of tuberculosis (HFS-TB). In parallel, we identified d-cycloserine MICs in 415 clinical Mycobacterium tuberculosis (Mtb) isolates from patients. We utilized these results, including intracavitary concentrations, to identify the clinical dose that would be able to achieve or exceed target exposures in 10000 patients using Monte Carlo experiments (MCEs). Results: There were no published d-cycloserine pharmacokinetics/pharmacodynamics studies identified. Therefore, we performed new HFS-TB experiments. Cyloserine killed 6.3 log10 colony-forming units (CFU)/mL extracellular bacilli over 28 days. Efficacy was driven by the percentage of time concentration persisted above MIC (%TMIC), with 1.0 log10 CFU/mL kill achieved by %TMIC = 30% (target exposure). The tentative epidemiological cutoff value with the Sensititre MYCOTB assay was 64 mg/L. In MCEs, 750 mg twice daily achieved target exposure in lung cavities of 92% of patients whereas 500 mg twice daily achieved target exposure in 85% of patients with meningitis. The proposed MCE-derived clinical susceptibility breakpoint at the proposed doses was 64 mg/L. Conclusions: Cycloserine is cidal against Mtb. The susceptibility breakpoint is 64 mg/L. However, the doses likely to achieve the cidality in patients are high, and could be neurotoxic.

The discovery of ceftazidime/avibactam as an anti-Mycobacterium avium agent.

OBJECTIVES: To determine if ceftaroline and ceftazidime combined with avibactam are efficacious against pulmonary Mycobacterium avium complex (MAC) disease. METHODS: First, we performed a concentration-effect study of ceftaroline and ceftaroline/avibactam against extracellular MAC in test tubes. Given the difficulty of obtaining avibactam at the time of experimentation, we used a single concentration of commercial ceftazidime/avibactam, and two sets of non-treated controls, one with ceftazidime/avibactam and the other without. After finding antimicrobial activity with the ceftazidime/avibactam 'control', we performed ceftazidime/avibactam dose-effect studies in test tubes against extracellular MAC and in 24-well plates against intracellular MAC. We then performed a ceftazidime/avibactam exposure-effect and dose-fractionation studies in the hollow-fibre system model of intracellular pulmonary MAC (HFS-MAC). In each experiment, we repetitively sampled each HFS-MAC at specified times to validate ceftazidime/avibactam pharmacokinetics and to quantify bacterial burden. RESULTS: Ceftaroline killed extracellular MAC with maximal microbial kill (Emax) of 4.87 ±âŸ0.26 log10 cfu/mL. However, the ceftazidime/avibactam 'control' also killed MAC compared with the non-treated control. Ceftazidime/avibactam Emax was 3.8 log10 cfu/mL against extracellular bacilli and 3.6 log10 cfu/mL against intracellular MAC. In the HFS-MAC, ceftazidime/avibactam achieved a half-life of 2.5-3.3 h and killed MAC 0.61-2.40 log10 cfu/mL below the starting bacterial burden. The ceftazidime/avibactam efficacy was linked to the proportion of the dosing interval for which the concentration persists above the MIC (fT>MIC), with optimal efficacy at free-drug fT>MIC of 52% (r2 = 0.95). CONCLUSIONS: Ceftazidime/avibactam effectively kills MAC at exposures easily achieved in the lung by clinical doses. Efficacy was higher than with clinically achievable doses of azithromycin and ethambutol.

Stimulation of unprimed macrophages with immune complexes triggers a low output of nitric oxide by calcium-dependent neuronal nitric-oxide synthase.

Immune complexes composed of IgG-opsonized pathogens, particles, or proteins are phagocytosed by macrophages through Fcγ receptors (FcγRs). Macrophages primed with IFNγ or other pro-inflammatory mediators respond to FcγR engagement by secreting high levels of cytokines and nitric oxide (NO). We found that unprimed macrophages produced lower levels of NO, which required efficient calcium (Ca(2+)) flux as demonstrated by using macrophages lacking selenoprotein K, which is required for FcγR-induced Ca(2+) flux. Thus, we further investigated the signaling pathways involved in low output NO and its functional significance. Evaluation of inducible, endothelial, and neuronal nitric-oxide synthases (iNOS, eNOS, and nNOS) revealed that FcγR stimulation in unprimed macrophages caused a marked Ca(2+)-dependent increase in both total and phosphorylated nNOS and slightly elevated levels of phosphorylated eNOS. Also activated were three MAP kinases, ERK, JNK, and p38, of which ERK activation was highly dependent on Ca(2+) flux. Inhibition of ERK reduced both nNOS activation and NO secretion. Finally, Transwell experiments showed that FcγR-induced NO functioned to increase the phagocytic capacity of other macrophages and required both NOS and ERK activity. The production of NO by macrophages is conventionally attributed to iNOS, but we have revealed an iNOS-independent receptor/enzyme system in unprimed macrophages that produces low output NO. Under these conditions, FcγR engagement relies on Ca(2+)-dependent ERK phosphorylation, which in turn increases nNOS and, to a lesser extent, eNOS, both of which produce low levels of NO that function to promote phagocytosis.

Human polyomavirus JC (JCV) infection of human B lymphocytes: a possible mechanism for JCV transmigration across the blood-brain barrier.

It has been suggested that JC virus (JCV) might travel to the central nervous system in infected B cells. Moreover, recent data suggest the presence of JCV in bone marrow plasma cells. However, the evidence for infection and replication of JCV in B cells is unclear. To address this question, we infected Epstein-Barr virus-transformed B cells with JCV and found that the viral genome decreased >1000-fold from days 0 to 20 after infection, which concurred with the absence of viral early and late messenger RNA transcripts and proteins. However, immunofluorescent images of B cells infected with fluorescein isothiocyanate-conjugated JCV demonstrated that JCV enters the B cells, and DNase protection assay confirmed the presence of intact JCV virions inside the B cells. Moreover, JCV-infected B cells were able to transmit infection to naive glial cells. These data confirm that JCV nonproductively infects B cells and possibly uses them as a vehicle for transmigration across the blood-brain barrier.

sncRNA-1 Is a Small Noncoding RNA Produced by Mycobacterium tuberculosis in Infected Cells That Positively Regulates Genes Coupled to Oleic Acid Biosynthesis.

Nearly one third of the world's population is infected with Mycobacterium tuberculosis (Mtb). While much work has focused on the role of different Mtb encoded proteins in pathogenesis, recent studies have revealed that Mtb also transcribes many noncoding RNAs whose functions remain poorly characterized. We performed RNA sequencing and identified a subset of Mtb H37Rv-encoded small RNAs (<30 nts in length) that were produced in infected macrophages. Designated as smaller noncoding RNAs (sncRNAs), three of these predominated the read counts. Each of the three, sncRNA-1, sncRNA-6, and sncRNA-8 had surrounding sequences with predicted stable secondary RNA stem loops. Site-directed mutagenesis of the precursor sequences suggest the existence of a hairpin loop dependent RNA processing mechanism. A functional assessment of sncRNA-1 suggested that it positively regulated two mycobacterial transcripts involved in oleic acid biosynthesis. Complementary loss- and gain- of-function approaches revealed that sncRNA-1 positively supports Mtb growth and survival in nutrient-depleted cultures as well as in infected macrophages. Overall, the findings reveal that Mtb produces sncRNAs in infected cells, with sncRNA-1 modulating mycobacterial gene expression including genes coupled to oleic acid biogenesis.

Serotonin receptor 2A blocker (risperidone) has no effect on human polyomavirus JC infection of primary human fetal glial cells.

A recent report demonstrated that JC virus (JCV) employs serotonin receptor 2A (5HT(2A)R) to infect the glial cells. To assess the ability of a potent 5HT(2A)R blocker, risperidone, to inhibit JCV infection, the authors treated primary human fetal glial (PHFG) cells in vitro with risperidone for 24 h and inoculated with JCV(Mad1). There was no significant difference in JCV genome copies or mRNA transcripts and protein expression in treatment-naive and risperidone-treated PHFG cells. These data indicate that risperidone does not inhibit JCV(Mad1) attachment, internalisation, and replication in PHFG cells, and 5HT(2A)R blockers may not be effective in treating progressive multifocal leukoencephalopathy (PML).

Comparison of real-time PCR and hemagglutination assay for quantitation of human polyomavirus JC.

Human polyomavirus JC (JCV), the etiological agent of the disease progressive multifocal leukoencephalopathy (PML) affects immunocompromised patients particularly patients with AIDS. In vitro studies of JCV infection are hampered by the lack of sensitive JCV quantitation tests. Although the hemagglutination (HA) assay has been routinely employed for in vitro quantitation of JCV, its sensitivity is severely limited. We have employed a real-time PCR assay which compares favorably with the HA assay for the in vitro quantitation of JCV. JCV(Mad1), propagated in primary human fetal glial (PHFG) cells in two independent laboratories, was purified and quantitated by the HA assay. Both batches of purified JCV(Mad1) were then serially diluted in Dulbecco's Modified Eagle's Medium to obtain HA titers ranging from 64 to 0.001 HA units (HAU) per 100 microL of virus suspension. DNA was extracted from 100 microL of virus suspension and eluted in 50 microL of buffer, and DNA amplification and quantitation were performed in the Bio-Rad iCycler iQ Multicolor Real-Time PCR Detection System using T-antigen as the target gene. Real-time PCR for quantitation of JCV was sensitive and consistently detected 1.8 x 10(1) copies of JCV DNA, and as low as 0.001 HAU equivalent of JCV. Moreover, there was a strong linear correlation between the HA assay and the DNA copy number of JCV(Mad1). The intra-run and inter-run coefficients of variation for the JCV standard curve were 0.06% to 4.8% and 2.6% to 5.2%, respectively. Based on these data, real-time PCR can replace the less-sensitive HA assay for the reliable detection, quantitation and monitoring of in vitro JCV replication.

Polyomavirus JC infects human brain microvascular endothelial cells independent of serotonin receptor 2A.

Although human polyomavirus JC (JCV) is known to cause progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals, the mechanism by which JCV crosses the blood-brain barrier (BBB) remains unclear. To test our hypothesis that cell-free JCV gains entry into the brain by infecting endothelial cells, we inoculated human brain microvascular endothelial (HBMVE) cells with 50 HAU (1.33+/-0.27 x 10(7) genome copies) of JCV(Mad1) and analyzed the expression of early and late viral genes and proteins by immunocytochemistry, quantitative real-time PCR (qPCR), quantitative real-time reverse transcriptase PCR (qRT-PCR) and immunoprecipitation followed by Western blotting. JCV infected and replicated efficiently in HBMVE cells and produced infectious virions several hundred fold higher than the infecting inoculum. HBMVE cells in vitro did not express serotonin receptor 2A (5HT(2A)R), and 5HT(2A)R blockers did not prevent JCV infection of HBMVE cells. Collectively, our data indicate that the productive in vitro infection of HBMVE cells by JCV is independent of 5HT(2A)R.