Sven O. Friedrich

ANG II type I receptor antagonism improved nitric oxide production and enhanced eNOS and PKB/Akt expression in hearts from a rat model of insulin resistance

Exogenous insulin therapy improves endothelial function in insulin resistant patients, indirectly indicating that nitric oxide synthase activity and NO production may be impaired. Insulin stimulates production of NO by activating a signaling pathway including insulin receptor substrate-1, phosphatidylinositol-3-kinase and protein kinase B (PKB/Akt). Angiotensin II type I (AT1) receptor-evoked oxidative stress is implicated in the inactivation of NO, impairing endothelium-dependent vasodilatation. Blocking the actions of Angiotensin II with an AT1 receptor antagonist (Losartan), has beneficial effects in patients with insulin resistance or type 2 diabetes mellitus. This study investigated whether elevated Angiotensin II influences myocardial insulin resistance, insulin signaling and NO production in a rat model of diet-induced obesity (DIO) by antagonizing the actions of the AT1 receptor with Losartan. Isolated, perfused hearts, Western blotting and flow-cytometric methods were utilized to determine myocardial function, expression and phosphorylation of key proteins and NO production, respectively. Results showed that hearts from DIO rats are insulin resistant (higher serine phosphorylation of IRS-1, lower insulin-stimulated phosphorylation of PKB/Akt and eNOS, lower NO production) and had poorer functional recovery and larger infarct development after ischaemia/reperfusion. Losartan improved the impaired functional recovery, and NO production and enhanced eNOS expression and phosphorylation and reduced infarct size in hearts from the DIO animals. Data obtained from Losartan treatment also revealed that Angiotensin II signaling modulates myocardial PKB/Akt expression. We conclude that Angiotensin II signaling exacerbates inhibition of NO production in insulin resistance and that this can be improved by AT1 antagonism.

Hypoxia-induced regulation of nitric oxide synthase in cardiac endothelial cells and myocytes and the role of the PI3-K/PKB pathway.

The roles of endothelial nitric oxide synthase (eNOS), and its putative association with protein kinase B (PKB), and inducible nitric oxide synthase (iNOS) are not well characterized in hypoxic cardiac cells and there is a lack of studies that measure nitric oxide (NO) directly. Objective To measure NO production in cardiomyocytes and cardiac microvascular endothelial cells (CMECs) under baseline and hypoxic conditions and to evaluate the expression, regulation and activation of eNOS, iNOS and PKB. The effect of PI3-K/PKB inhibition on NO production and eNOS expression/activation was also investigated. Methods Adult rat cardiomyocytes and rat CMECs were made hypoxic by cell pelleting and low PO2 incubation. Intracellular NO was measured by FACS analysis of DAF-2/DA fluorescence, and eNOS, iNOS and PKB were evaluated by Western blotting or flow cytometry. Upstream PKB inhibition was achieved with wortmannin. Results (1) NO levels increased in both cell types after exposure to hypoxia. (2) In hypoxic CMECs, eNOS was upregulated and activated, no iNOS expression was observed and PKB was activated. (3) In myocytes, hypoxia did not affect eNOS expression, but increased its activation. Activated PKB also increased during hypoxia. FACS analysis showed increased iNOS in hypoxic myocytes. (4) Wortmannin resulted in decreased hypoxia-induced NO production and reduced activated eNOS levels. Conclusions Cardiomyocytes and CMECs show increased NO production during hypoxia. eNOS seems to be the main NOS isoform involved as source of the increased NO generation, although there may be a role for iNOS and other non-eNOS sources of NO in the hypoxic myocytes. Hypoxia-induced PKB and eNOS activation occurred simultaneously in both cell types, and the PI3-K/PKB pathway was associated with hypoxia-induced NO production via eNOS activation.

Simultaneous staining of sputum smears for acid-fast and lipid-containing Myobacterium tuberculosis can enhance the clinical evaluation of antituberculosis treatments

Dormant, slow-growing, antibiotic-tolerant Mycobacterium tuberculosis undermine the shortening of tuberculosis treatment to less than 6 months and are thought to be characterised by intracellular lipid bodies. Antibiotic effects on such persisting bacilli escape evaluation as they cannot be readily cultured. We identified cells containing lipid bodies in sputum smears from 86 newly diagnosed pulmonary tuberculosis patients and monitored these cells daily in 42 patients over the first 14 days of treatment with rifampicin, the experimental compound SQ-109, or both agents combined. Counts of Nile-Red-positive lipid-body containing cells were correlated with those of Auramine-O-positive cells and colony forming units of viable Mycobacterium tuberculosis on agar plates. Rifampicin but not SQ-109 significantly reduced colony forming units but all treatments distinctively and significantly changed the proportions of lipid body-containing bacilli and viable Mycobacterium tuberculosis. Monitoring lipid-body containing bacilli in sputum during treatment with experimental antituberculosis regimens may identify putative treatment-shortening regimens.

Xpert MTB/RIF on Stool is Useful for the Rapid Diagnosis of Tuberculosis in Young Children with Severe Pulmonary Disease.

Background: Tuberculosis (TB) continues to result in high morbidity and mortality in children from resource-limited settings. Diagnostic challenges, including resource-intense sputum collection methods and insensitive diagnostic tests, contribute to diagnostic delay and poor outcomes in children. We evaluated the diagnostic utility of stool Xpert MTB/RIF (Xpert) compared with bacteriologic confirmation (combination of Xpert and culture of respiratory samples). Methods: In a hospital-based study in Cape Town, South Africa, we enrolled children younger than 13 years of age with suspected pulmonary TB from April 2012 to August 2015. Standard clinical investigations included tuberculin skin test, chest radiograph and HIV testing. Respiratory samples for smear microscopy, Xpert and liquid culture included gastric aspirates, induced sputum, nasopharyngeal aspirates and expectorated sputum. One stool sample per child was collected and tested using Xpert. Results: Of 379 children enrolled (median age, 15.9 months, 13.7% HIV infected), 73 (19.3%) had bacteriologically confirmed TB. The sensitivity and specificity of stool Xpert versus overall bacteriologic confirmation were 31.9% [95% confidence interval (CI): 21.84%–44.50%] and 99.7% (95% CI: 98.2%–100%), respectively. A total of 23/51 (45.1%) children with bacteriologically confirmed TB with severe disease were stool Xpert positive. Cavities on chest radiograph were associated with Xpert stool positivity regardless of age and other relevant factors [odds ratios (OR) 7.05; 95% CI: 2.16–22.98; P = 0.001]. Conclusions: Stool Xpert can rapidly confirm TB in children who present with radiologic findings suggestive of severe TB. In resource-limited settings where children frequently present with advanced disease, Xpert on stool samples could improve access to rapid diagnostic confirmation and appropriate treatment.

Xpert MTB/RIF Assay Shows Faster Clearance of Mycobacterium tuberculosis DNA with Higher Levels of Rifapentine Exposure

ABSTRACT The Xpert MTB/RIF assay is both sensitive and specific as a diagnostic test. Xpert also reports quantitative output in cycle threshold (CT ) values, which may provide a dynamic measure of sputum bacillary burden when used longitudinally. We evaluated the relationship between Xpert CT trajectory and drug exposure during tuberculosis (TB) treatment to assess the potential utility of Xpert CT for treatment monitoring. We obtained serial sputum samples from patients with smear-positive pulmonary TB who were consecutively enrolled at 10 international clinical trial sites participating in study 29X, a CDC-sponsored Tuberculosis Trials Consortium study evaluating the tolerability, safety, and antimicrobial activity of rifapentine at daily doses of up to 20 mg/kg of body weight. Xpert was performed at weeks 0, 2, 4, 6, 8, and 12. Longitudinal CT data were modeled using a nonlinear mixed effects model in relation to rifapentine exposure (area under the concentration-time curve [AUC]). The rate of change of CT was higher in subjects receiving rifapentine than in subjects receiving standard-dose rifampin. Moreover, rifapentine exposure, but not assigned dose, was significantly associated with rate of change in CT (P = 0.02). The estimated increase in CT slope for every additional 100 μg · h/ml of rifapentine drug exposure (as measured by AUC) was 0.11 CT /week (95% confidence interval [CI], 0.05 to 0.17). Increasing rifapentine exposure is associated with a higher rate of change of Xpert CT , indicating faster clearance of Mycobacterium tuberculosis DNA. These data suggest that the quantitative outputs of the Xpert MTB/RIF assay may be useful as a dynamic measure of TB treatment response.

Sputum Volume Predicts Sputum Mycobacterial Load during the First 2 Weeks of Antituberculosis Treatment

ABSTRACT Disease severity in patients with pulmonary tuberculosis is associated with mycobacterial sputum load. To ascertain whether reduced sputum production during treatment is a useful clinical sign of improvement, we analyzed the mycobacterial loads of 5,552 sputum samples collected from 439 newly diagnosed sputum smear-positive tuberculosis patients who participated in six 14-day studies of antituberculosis treatment. Sputum volumes were categorized as low (<6 ml), medium (6 to 10 ml), or large (>10 ml), and mycobacterial load was measured by the time to positivity in liquid culture and the CFU counts on solid culture. The association of sputum volume with mycobacterial load was estimated with multiple linear regression models adjusted for repeated measures. The predictor variables were sputum volume category, treatment day, specific study , and the interaction of sputum volume category and treatment day. Mycobacterial load was significantly associated only with the day on treatment and sputum volume, which tended to decrease with ongoing treatment. With the volume held constant, each day on treatment decreased the log CFU by 0.082 (P < 0.001) and increased the time to positivity (TTP) by 1.04 h (P < 0.001). From low to medium and from medium to large sputum volumes, the log CFU/ml increased by 0.265 (P < 0.003) and 0.490 (P < 0.001), respectively, and the TTP decreased by 1.17 h (P < 0.001) and 1.30 h (P < 0.001), respectively, for a given day of treatment. The variability of the sputum load measurements increased with the day of treatment and lower sputum volumes. The significant association of sputum volume and mycobacterial load validates decreasing sputum production as a clinical sign of improvement during early antituberculosis treatment.

Short-Term Storage Does Not Affect the Quantitative Yield of Mycobacterium tuberculosis in Sputum in Early-Bactericidal-Activity Studies

ABSTRACT Early-bactericidal-activity (EBA) studies measure the change in mycobacterial load in sputum over time to evaluate antituberculosis drugs. We investigated whether a delay in sputum processing influences the quantitative results of sputum mycobacterial culture. We identified pretreatment smear-positive sputum samples collected overnight and processed at a single laboratory. Sputum volume, time from sputum collection to processing, CFU counts/ml of sputum, and time to culture positivity (TTP) data were retrieved. We obtained 817 TTP and 794 CFU results from a total of 844 sputum samples. Contamination did not occur more frequently with prolonged storage (TTP, 2.0%; CFU, 2.4%). Sample volumes were <5 ml in 5%, 5 to 10 ml in 46%, and >10 ml in 49%. Delays to processing were 0, 1, 2, and 3 days in 696 (43.2%), 722 (44.8%), 128 (7.9%), and 65 (4.0%) samples, respectively. TTP and CFU did not significantly differ between days of delay to processing (P = 0.098 and P = 0.908, respectively), but there was a nonsignificant trend toward a prolonged TTP over time (P = 0.052, Jonckheere-Terpstra trend test). Sputa of <5 ml in volume showed a significantly prolonged TTP compared to sputum of >5 ml (113 h versus 99 h; P < 0.01) but no significant decrease in CFU. Sputum can be stored under refrigerated conditions for deferred processing for at least 3 days. This means that central laboratories can be used for quantitative mycobacterial study endpoints when delays to processing are not expected to exceed a few days. Care should be taken to collect sputum of sufficient volume.

Acquisition of Rifampin Resistance in Pulmonary Tuberculosis

ABSTRACT Mycobacterium tuberculosis strains with spontaneous mutations conferring resistance to rifampin (RIF) are exceedingly rare, and fixed drug combinations typically prevent augmentation of resistance to single drugs. Fourteen newly diagnosed tuberculosis patients were treated with RIF alone for 14 days, and bacterial loads, including mutation frequencies, were determined. A statistical model estimated that 1% of the remaining viable mycobacteria could be RIF resistant after 30 days of monotherapy. This indicates that temporal and spatial windows of RIF monotherapy due to uneven drug distribution within lung lesions could contribute to the acquisition of resistance to RIF.