Determining the Delamanid Pharmacokinetics/Pharmacodynamics Susceptibility Breakpoint Using Monte Carlo Experiments.

Antimicrobial susceptibility testing, based on clinical breakpoints that incorporate pharmacokinetics/pharmacodynamics (PK/PD) and clinical outcomes, is becoming a new standard in guiding individual patient therapy as well as for drug resistance surveillance. However, for most antituberculosis drugs, breakpoints are instead defined by the epidemiological cutoff values of the MIC of phenotypically wild-type strains irrespective of PK/PD or dose. In this study, we determined the PK/PD breakpoint for delamanid by estimating the probability of target attainment for the approved dose administered at 100 mg twice daily using Monte Carlo experiments. We used the PK/PD targets (0- to 24-h area under the concentration-time curve to MIC) identified in a murine chronic tuberculosis model, hollow fiber system model of tuberculosis, early bactericidal activity studies of patients with drug-susceptible tuberculosis, and population pharmacokinetics in patients with tuberculosis. At the MIC of 0.016 mg/L, determined using Middlebrook 7H11 agar, the probability of target attainment was 100% in the 10,000 simulated subjects. The probability of target attainment fell to 25%, 40%, and 68% for PK/PD targets derived from the mouse model, the hollow fiber system model of tuberculosis, and patients, respectively, at the MIC of 0.031 mg/L. This indicates that an MIC of 0.016 mg/L is the delamanid PK/PD breakpoint for delamanid at 100 mg twice daily. Our study demonstrated that it is feasible to use PK/PD approaches to define a breakpoint for an antituberculosis drug.

Phase I Single Ascending Dose and Food Effect Study in Healthy Adults and Phase I/IIa Multiple Ascending Dose Study in Patients with Pulmonary Tuberculosis to Assess Pharmacokinetics, Bactericidal Activity, Tolerability, and Safety of OPC-167832.

OPC-167832, an inhibitor of decaprenylphosphoryl-ß-d-ribose 2'-oxidase, demonstrated potent antituberculosis activity and a favorable safety profile in preclinical studies. This report describes the first two clinical studies of OPC-167832: (i) a phase I single ascending dose (SAD) and food effects study in healthy participants; and (ii) a 14-day phase I/IIa multiple ascending dose (MAD; 3/10/30/90 mg QD) and early bactericidal activity (EBA) trial in participants with drug-susceptible pulmonary tuberculosis (TB). OPC-167832 was well tolerated at single ascending doses (10 to 480 mg) in healthy participants and multiple ascending doses (3 to 90 mg) in participants with TB. In both populations, nearly all treatment-related adverse events were mild and self-limiting, with headache and pruritus being the most common events. Abnormal electrocardiograms results were rare and clinically insignificant. In the MAD study, OPC-167832 plasma exposure increased in a less than dose-proportional manner, with mean accumulation ratios ranging from 1.26 to 1.56 for Cmax and 1.55 to 2.01 for area under the concentration-time curve from 0 to 24 h (AUC0-24h). Mean terminal half-lives ranged from 15.1 to 23.6 h. Pharmacokinetics (PK) characteristics were comparable to healthy participants. In the food effects study, PK exposure increased by less than ~2-fold under fed conditions compared to the fasted state; minimal differences were observed between standard and high-fat meals. Once-daily OPC-167832 showed 14-day bactericidal activity from 3 mg (log10 CFU mean ± standard deviation change from baseline; -1.69 ± 1.15) to 90 mg (-2.08 ± 0.75), while the EBA of Rifafour e-275 was -2.79 ± 0.96. OPC-167832 demonstrated favorable pharmacokinetic and safety profiles, as well as potent EBA in participants with drug-susceptible pulmonary TB.

Ultrasensitive detection of CA125 based on a triple signal amplification strategy with a huge number of loaded probes via exonuclease cyclic cleavage, rolling cyclic amplification and strand self-growth.

A novel electrochemiluminescence (ECL) aptamer biosensor with high sensitivity and selectivity for the detection of tumor biomarker carbohydrate antigen 125 (CA125) was constructed, and a strategy of triple amplification of signals was proposed using an exonuclease cyclic cleavage aptamer, combined with rolling ring amplification technologies, generating multi-branched dendritic double-stranded DNA to load a large number of probes through "strand self-growth". The double-stranded DNA, which is abbreviated as CP/CA dsDNA, formed by hybridizing the single strand of capture DNA (CP DNA) with the single strand DNA of the CA125 aptamer (CA Apt) was modified on Fe3O4@Au. When CA125 was added, CP/CA dsDNA was unwound, and CA125 specifically combined with CA Apt to form a protein-aptamer complex, leaving only CP DNA on the surface of Fe3O4@Au. RecJf exonuclease cleaved the aptamer in the protein-aptamer complex and released CA125, which recombined with other CA125 aptamers, to form a cycle that produces more CP DNA on Fe3O4@Au. Three ssDNA (H1, H2, and H3) were introduced and hybridized with CP DNA to form a dsDNA with a "+" configuration structure. Then phi29 DNA polymerase, T4 DNA ligase, deoxy-ribonucleoside triphosphate (dNTP) and padlock probes were added to form a large number of complementary strands of padlock probes (CS padlock probes) by rolling cyclic amplification. CS padlock probes were linked to the "+" type dsDNA; then ssDNA H4 was added and hybridized with the CS padlock probe to form multi-branched dendritic dsDNA. A large number of tris(2,2'-bipyridyl)ruthenium(II) probes were embedded in the double strands, resulting in an extremely strong ECL signal in the presence of the co-reactant tri-n-propylamine (TPA). There is a linear relationship between the ECL signals and CA125 concentrations in the range of 1.0 × 10-15-1.0 × 10-8 mg mL-1, and the detection limit was 2.38 × 10-16 mg mL-1. It has been used for the determination of CA125 in serum samples.

Sputum lipoarabinomannan (LAM) as a biomarker to determine sputum mycobacterial load: exploratory and model-based analyses of integrated data from four cohorts.

BACKGROUND: Despite the high global disease burden of tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb) infection, novel treatments remain an urgent medical need. Development efforts continue to be hampered by the reliance on culture-based methods, which often take weeks to obtain due to the slow growth rate of Mtb. The availability of a "real-time" measure of treatment efficacy could accelerate TB drug development. Sputum lipoarabinomannan (LAM; an Mtb cell wall glycolipid) has promise as a pharmacodynamic biomarker of mycobacterial sputum load. METHODS: The present analysis evaluates LAM as a surrogate for Mtb burden in the sputum samples from 4 cohorts of a total of 776 participants. These include those from 2 cohorts of 558 non-TB and TB participants prior to the initiation of treatment (558 sputum samples), 1 cohort of 178 TB patients under a 14-day bactericidal activity trial with various mono- or multi-TB drug therapies, and 1 cohort of 40 TB patients with data from the first 56-day treatment of a standard 4-drug regimen. RESULTS: Regression analysis demonstrated that LAM was a predictor of colony-forming unit (CFU)/mL values obtained from the 14-day treatment cohort, with well-estimated model parameters (relative standard error ≤ 22.2%). Moreover, no changes in the relationship between LAM and CFU/mL were observed across the different treatments, suggesting that sputum LAM can be used to reasonably estimate the CFU/mL in the presence of treatment. The integrated analysis showed that sputum LAM also appears to be as good a predictor of time to Mycobacteria Growth Incubator Tube (MGIT) positivity as CFU/mL. As a binary readout, sputum LAM positivity is a strong predictor of solid media or MGIT culture positivity with an area-under-the-curve value of 0.979 and 0.976, respectively, from receiver-operator curve analysis. CONCLUSIONS: Our results indicate that sputum LAM performs as a pharmacodynamic biomarker for rapid measurement of Mtb burden in sputum, and thereby may enable more efficient early phase clinical trial designs (e.g., adaptive designs) to compare candidate anti-TB regimens and streamline dose selection for use in pivotal trials. Trial registration NexGen EBA study (NCT02371681).

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.

Factors related to disagreement between self-reported versus objective measurement of allergen sensitization at a tertiary pediatric center in Beijing, China.

BACKGROUND: The objectives of present study were to examine the level of disagreement between self-reported and objective measurement of sensitization to common inhalant allergens, and to explore the potential risk factors that might contribute to this discrepancy. METHODS: A total of 215 children were enrolled from pediatric clinics at a tertiary pediatric center in Beijing, China. A survey questionnaires regarding self-perceived sensitization was completed by participants' parents/caregiver, meanwhile, skin prick testing(SPT) was performed as objective assessment of sensitization. Extent of agreement between self-reported versus SPT-measured sensitization to individual allergen was calculated using Cohen's kappa (κ) coefficient. Multivariable regression analyses were used to determine the factors associated with discrepancy between self-reported and objective measurement of sensitization. RESULTS: 119(55.3%) patients have reported to be sensitized to at least one of inhalant allergen, whereas 167(77.7%) patients had a positive skin testing response. Agreement between self-perceived and actual aeroallergen sensitization was moderate for mites(κ = 0.518) and grass pollen mix(κ = 0.451), moreover, fair agreement was observed for mold(κ = 0.316) and cockroach(κ = 0.297), respectively. There was a least agreement between perceived and actual sensitization observed for pet dander, with a kappa coefficient of 0.005. Subjects' age, atopy history, ownership of pet may increase the risk of disagreement, moreover, background factors of informant, like: age, education level, and the relationship with enrolled subjects, were linked to the incidence of disagreement between self-reported sensitization in comparison with SPT results. CONCLUSION: Questionnaire-based self-assessment is easy way to collect clinical information on allergen sensitization; however, the accuracy of questionnaire-derived information is more likely to be influenced by respondent's background factors. The information from the questionnaire report is considered to be more reliable when in combination with objective assessment of sensitization, including blood IgE testing and SPT.

Lipoarabinomannan in sputum to detect bacterial load and treatment response in patients with pulmonary tuberculosis: Analytic validation and evaluation in two cohorts.

BACKGROUND: Lipoarabinomannan (LAM) is a major antigen of Mycobacterium tuberculosis (MTB). In this report, we evaluated the ability of a novel immunoassay to measure concentrations of LAM in sputum as a biomarker of bacterial load prior to and during treatment in pulmonary tuberculosis (TB) patients. METHODS AND FINDINGS: Phage display technology was used to isolate monoclonal antibodies binding to epitopes unique in LAM from MTB and slow-growing nontuberculous mycobacteria (NTM). Using these antibodies, a sandwich enzyme-linked immunosorbent assay (LAM-ELISA) was developed to quantitate LAM concentration. The LAM-ELISA had a lower limit of quantification of 15 pg/mL LAM, corresponding to 121 colony-forming units (CFUs)/mL of MTB strain H37Rv. It detected slow-growing NTMs but without cross-reacting to common oral bacteria. Two clinical studies were performed between the years 2013 and 2016 in Manila, Philippines, in patients without known human immunodeficiency virus (HIV) coinfection. In a case-control cohort diagnostic study, sputum specimens were collected from 308 patients (aged 17-69 years; 62% male) diagnosed as having pulmonary TB diseases or non-TB diseases, but who could expectorate sputum, and were then evaluated by smear microscopy, BACTEC MGIT 960 Mycobacterial Detection System (MGIT) and Lowenstein-Jensen (LJ) culture, and LAM-ELISA. Some sputum specimens were also examined by Xpert MTB/RIF. The LAM-ELISA detected all smear- and MTB-culture-positive samples (n = 70) and 50% (n = 29) of smear-negative but culture-positive samples (n = 58) (versus 79.3%; 46 positive cases by the Xpert MTB/RIF), but none from non-TB patients (n = 56). Among both LAM and MGIT MTB-culture-positive samples, log10-transformed LAM concentration and MGIT time to detection (TTD) showed a good inverse relationship (r = -0.803, p < 0.0001). In a prospective longitudinal cohort study, 40 drug-susceptible pulmonary TB patients (aged 18-69 years; 60% male) were enrolled during the first 56 days of the standard 4-drug therapy. Declines in sputum LAM concentrations correlated with increases of MGIT TTD in individual patients. There was a 1.29 log10 decrease of sputum LAM concentration, corresponding to an increase of 221 hours for MGIT TTD during the first 14 days of treatment, a treatment duration often used in early bactericidal activity (EBA) trials. Major limitations of this study include a relatively small number of patients, treatment duration up to only 56 days, lack of quantitative sputum culture CFU count data, and no examination of the correlation of sputum LAM to clinical cure. CONCLUSIONS: These results indicate that the LAM-ELISA can determine LAM concentration in sputum, and sputum LAM measured by the assay may be used as a biomarker of bacterial load prior to and during TB treatment. Additional studies are needed to examine the predictive value of this novel biomarker on treatment outcomes.

MIC of Delamanid (OPC-67683) against Mycobacterium tuberculosis Clinical Isolates and a Proposed Critical Concentration.

The increasing global burden of multidrug-resistant tuberculosis (MDR-TB) requires reliable drug susceptibility testing that accurately characterizes susceptibility and resistance of pathogenic bacteria to effectively treat patients with this deadly disease. Delamanid is an anti-TB agent first approved in the European Union in 2014 for the treatment of pulmonary MDR-TB in adults. Using the agar proportion method, delamanid MIC was determined for 460 isolates: 316 from patients enrolled in a phase 2 global clinical trial, 76 from two phase 2 early bactericidal activity trials conducted in South Africa, and 68 isolates obtained outside clinical trials (45 from Japanese patients and 23 from South African patients). With the exception of two isolates, MICs ranged from 0.001 to 0.05 µg/ml, resulting in an MIC50 of 0.004 µg/ml and an MIC90 of 0.012 µg/ml. Various degrees of resistance to other anti-TB drugs did not affect the distribution of MICs, nor did origin of isolates from regions/countries other than South Africa. A critical concentration/breakpoint of 0.2 µg/ml can be used to define susceptible and resistant isolates based on the distribution of MICs and available pharmacokinetic data. Thus, clinical isolates from delamanid-naive patients with tuberculosis have a very low MIC for delamanid and baseline resistance is rare, demonstrating the potential potency of delamanid and supporting its use in an optimized background treatment regimen for MDR-TB.

[Quality control for maximal expiratory flow-volume curve as a pulmonary function test in school-age children].

OBJECTIVE: To assess the quality control for the maximal expiratory flow-volume (MEFV) curve in school-age children. METHODS: Eight hundred and sixty-two children who had two or more MEFV manoeuvres were classified into ≥6-year-old (n=379), ≥8-year-old (n=210), ≥10-year-old (n=64), and 12-17-year-old groups (n=109). The parameters of quality control and concordance with quality control criteria for MEFV were compared between the two groups. In addition, patients who were diagnosed with asthma were classified into two groups, one with normal pulmonary function (n=155) and the other with abnormal pulmonary function (n=62), based on the results of spirometry. Differences in the parameters of quality control for spirometry were compared between the two groups. RESULTS: Eight hundred and sixty-two children underwent 2 367 MEFV manoeuvres, 97.8% of which met the start of test criterion for backward extrapolated volume (VBE) of less than 0.15 L, with the highest concordance in the ≥6-year-old group and the lowest concordance in the 12-17-year-old group. Three hundred and eighty-one children (44.2%) met the end of test criterion for forced expiratory time (FET) and the concordance in children over 10 years of age was lower than that in children under 10 years of age (P<0.05). Differences in two best forced expiratory volume in first second (FEV1) and forced vital capacity (FVC) manoeuvres were within 150 mL in 91.9% and 84.8%, respectively, of the children. The parameters of quality control for spirometry were better for asthmatic children with abnormal pulmonary function compared with asthmatic children with normal pulmonary function (P<0.05). CONCLUSIONS: Concordance with the start of test criteria and the manoeuvre repeatability criteria is high, whereas the concordance with the end of test criteria is low. It is suggested that the concordance with the FET criteria should be improved.

[Related factors for asthmatic children's responses to long-term treatment].

OBJECTIVE: To study the changes in pulmonary function and fractional exhaled nitric oxide in exhaled breath (FeNO) in asthmatic children who have different responses to regular treatment. METHODS: A total of 52 asthmatic children who had a good compliance with regular stepped control treatment were selected as subjects. They were followed up every three months to evaluate the asthma control level, pulmonary ventilation function, and FeNO for 9 months. Besides, medications for asthma control were recorded. RESULTS: At three follow-up points (months 3, 6, and 9), the percentage of asthmatic children who used the first or the second level of control treatment in the stable group (with stable response to the treatment) was significantly higher than in the unstable group (with unstable response to the treatment) (P<0.05), while the percentage of asthmatic children who used the third level of control treatment in the stable group was significantly lower than in the unstable group (P<0.05). At the three follow-up points, the stable group had a significantly higher ratio of forced expiratory volume in 1 second to forced vital capacity (FEV1/FVC) than the unstable group (P<0.05); at the 3-month and 9-month follow-up points, the stable group had a significantly higher percentage of predicted maximum mid-expiratory flow (MMEF%) than the unstable group (P<0.05); at the initial evaluation and 3-month follow-up point, the stable group had a significantly higher FeNO than the unstable group (P<0.05). CONCLUSIONS: Continuously monitoring FEV1/FVC, MMEF% and FeNO is useful in the early evaluation of the responses to treatment in children with asthma.

[A comparision of the effects of subcutaneous and sublingual immunotherapy on immunological responses in children with asthma].

OBJECTIVE: To compare the difference in the effects of subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) on immunological responses in children with asthma. METHODS: A total of 86 children with asthma caused by dust mites were enrolled and divided into a SLIT group (n=29), a SCIT group (n=13), a group receiving complete SCIT course (complete SCIT group; n=14), and a group receiving conventional medication (control group, n=30). Peripheral blood mononuclear cells were isolated and stimulated with house dust mite extract for 48 hours in vitro, and the percentage of regulatory T cells (Treg%) in CD4+ T cells was measured by flow cytometry. Analysis of variance with repeated measures was applied to compare the changes in humoral immunological indices and therapeutic effects in the SCIT and SLIT groups before treatment and after 6 and 12 months of treatment. RESULTS: Before antigenic stimulation, Treg% in CD4(+) T cells in the SCIT group was significantly higher than that in the SLIT and control groups; after antigenic stimulation was given, Treg% in the four groups decreased significantly. After 6 and 12 months of immunotherapy, the SCIT group had significant changes in serum sIgE and sIgG4 levels, while the SLIT group only showed a significant change in serum sIgE level. CONCLUSIONS: Temporal difference exists in different immunotherapies to cause immunological responses in children with asthma, and immunological responses induced by SCIT may occur earlier.

A simple and effective convolutional operator for node classification without features by graph convolutional networks.

Graph neural networks (GNNs), with their ability to incorporate node features into graph learning, have achieved impressive performance in many graph analysis tasks. However, current GNNs including the popular graph convolutional network (GCN) cannot obtain competitive results on the graphs without node features. In this work, we first introduce path-driven neighborhoods, and then define an extensional adjacency matrix as a convolutional operator. Second, we propose an approach named exopGCN which integrates the simple and effective convolutional operator into GCN to classify the nodes in the graphs without features. Experiments on six real-world graphs without node features indicate that exopGCN achieves better performance than other GNNs on node classification. Furthermore, by adding the simple convolutional operator into 13 GNNs, the accuracy of these methods are improved remarkably, which means that our research can offer a general skill to improve accuracy of GNNs. More importantly, we study the relationship between node classification by GCN without node features and community detection. Extensive experiments including six real-world graphs and nine synthetic graphs demonstrate that the positive relationship between them can provide a new direction on exploring the theories of GCNs.

Quabodepistat in combination with delamanid and bedaquiline in participants with drug-susceptible pulmonary tuberculosis: protocol for a multicenter, phase 2b/c, open-label, randomized, dose-finding trial to evaluate safety and efficacy.

BACKGROUND: Delamanid and bedaquiline are two of the most recently developed antituberculosis (TB) drugs that have been extensively studied in patients with multidrug-resistant TB. There is currently a need for more potent, less-toxic drugs with novel mechanisms of action that can be used in combination with these newer agents to shorten the duration of treatment as well as prevent the development of drug resistance. Quabodepistat (QBS) is a newly discovered inhibitor of decaprenylphosphoryl-ß-D-ribose-2'-oxidase, an essential enzyme for Mycobacterium tuberculosis to synthesize key components of its cell wall. The objective of this study is to evaluate the safety, efficacy, and appropriate dosing of a 4-month regimen of QBS in combination with delamanid and bedaquiline in participants with drug-susceptible pulmonary TB in comparison with the 6-month standard treatment (i.e., rifampicin, isoniazid, ethambutol, and pyrazinamide). METHODS: This phase 2b/c, open-label, randomized, parallel group, dose-finding trial will enroll approximately 120 participants (including no more than 15% with human immunodeficiency virus [HIV] coinfection) aged ≥ 18 to ≤ 65 years at screening with newly diagnosed pulmonary drug-sensitive TB from ~8 sites in South Africa. Following a screening period of up to 14 days, eligible participants will be randomized in a ratio of 1:2:2:1 to one of four arms. Randomization will be stratified by HIV status and the presence of bilateral cavitation on a screening chest x-ray. After the end of the treatment period, participants will be followed until 12 months post randomization. The primary efficacy endpoint is the proportion of participants achieving sputum culture conversion in Mycobacteria Growth Indicator Tube by the end of the treatment period. The safety endpoints consist of adverse events, clinical laboratory tests, vital signs, physical examination findings, and electrocardiographic changes. DISCUSSION: QBS's potent bactericidal activity and distinct mechanism of action (compared with other TB drugs currently available for human use) may make it an ideal candidate for inclusion in a novel treatment regimen to improve efficacy and potentially prevent resistance to concomitant TB drugs. This trial will assess the effectiveness, safety, and dosing of a new, shorter, QBS-based, combination anti-TB treatment regimen. TRIAL STATUS: ClinicalTrials.gov NCT05221502. Registered on February 3, 2022.

Two classes of anti-platelet drugs reduce anatomical infarct size in monkey hearts.

BACKGROUND: Recent studies in rabbits have demonstrated that platelet P2Y12 receptor antagonists are cardioprotective, and that the mechanism is surprisingly not related to blockade of platelet aggregation but rather to triggering of the same signal transduction pathway seen in pre- and postconditioning. We wanted to determine whether this same cardioprotection could be documented in a primate model and whether the protection was limited to P2Y12 receptor antagonists or was a class effect. METHODS: Thirty-one macaque monkeys underwent 90-min LAD occlusion/4-h reperfusion. RESULTS: The platelet P2Y12 receptor blocker cangrelor started just prior to reperfusion significantly decreased infarction by an amount equivalent to that seen with ischemic postconditioning (p < 0.001). For any size of risk zone, infarct size in treated hearts was significantly smaller than that in control hearts. OM2, an investigational murine antibody against the primate collagen receptor glycoprotein (GP) VI, produced similar protection (p < 0.01) suggesting a class effect. Both cangrelor and OM2 were quite effective at blocking platelet aggregation (94 % and 97 %, respectively). CONCLUSIONS: Thus in a primate model in which infarct size could be determined directly platelet anti-aggregatory agents are cardioprotective. The important implication of these investigations is that patients with acute myocardial infarction who are treated with platelet anti-aggregatory agents prior to revascularization may already be in a postconditioned state. This hypothesis may explain why in recent clinical trials postconditioning-mimetic interventions which were so protective in animal models had at best only a modest effect.

Phosphodiesterases as targets for intermittent claudication.

Intermittent claudication (IC) is one of the most frequent forms of lower extremity peripheral arterial disease (PAD) and is most commonly caused by arterial atherosclerosis. Its clinical manifestation includes fatigue, discomfort, or pain occurring in limb muscles due to exercise-induced ischemia, thus limiting the ability of IC patients to walk and exercise. In addition to lifestyle changes (diet, exercise, and smoking cessation), pharmacological treatments are needed. Pathologically, atherosclerotic lesions cause a mismatch in oxygen supply and metabolic demand in the leg muscles during walking/exercise. This subjects the muscles to repeated ischemia and reperfusion injury that can alter structure and oxidative metabolism, resulting in insufficient utilization of oxygen supply. Despite extensive research efforts, cilostazol and pentoxifylline are the only drugs indicated for relieving the symptoms of IC, with cilostazol demonstrating significant improvement in walking distance and quality of life in these patients. Originally developed as a PDE3 inhibitor, cilostazol was later found to have several other pharmacological actions, and its success has been attributed to its multifactorial actions on platelets, endothelium, smooth muscle, and lipid profiles. Using cilostazol as an example, we discuss the rationales and pitfalls of targeting PDEs in IC, and potential strategies for the development of new and more effective pharmacological treatments.

Attenuation of infarction in cynomolgus monkeys: preconditioning and postconditioning.

Ischemic pre- (IPC) and post- (IPOC) conditioning are very protective in laboratory animals, but it has not been possible to measure their anti-infarct potency in human hearts. Non-human primates are genetically closer to humans than other laboratory animals, but until now there have been no studies of IPC or IPOC in any primate species. Accordingly the left anterior descending coronary artery of cynomolgus monkeys was occluded for 90 min and reperfused for 4 h. In control animals, only 44% of the risk zone infarcted indicating cynomolgus myocardium is much more resistant to infarction than that of rabbits or rats. The regression line for the infarct-risk zone plot was very linear (r = 0.99), and intersected the risk zone axis at 0.82 cm3. Even small changes in infarct size could be detected as a shift in this line. Collateral flow in 12 monkeys was 6.6% of flow to normal myocardium and not a covariate of infarct size. IPC with two cycles of 10-min coronary occlusion/10-min reperfusion reduced infarction to near zero indicating that the innate resistance to infarction was not caused by constitutive preconditioning. Wortmannin, an antagonist of phosphatidylinositol 3-kinase (PI3-K), administered just before release of the 90-min coronary occlusion attenuated IPC's infarct-sparing effect by approximately 50% suggesting that PI3-K was involved in preconditioning's protection. IPOC with six cycles of 30-s reperfusion/30-s coronary reocclusion, a very protective protocol in most species, was much less protective than IPC. We conclude that ischemic preconditioning is extremely protective in cynomolgus hearts despite their sparse collateralization but, surprisingly, the protocol of IPOC used in this study offers less protection.

Induction of CD4+CD25+Foxp3+IL-10+ T cells in HDM-allergic asthmatic children with or without SIT.

BACKGROUND: Regulatory T cells and immunosuppressive cytokines, such as IL-10 and TGF-beta(1), may have a role in clinically effective allergen-specific immunotherapy. IL-10-secreting regulatory T cells have emerged as potential mediators of immune tolerance in numerous murine models of immunopathology. The aim of this study was to evaluate the frequency and function of regulatory T cells in the response to house dust mite (HDM) immunotherapy. METHODS: PBMCs were isolated from 27 HDM-allergic asthmatic children who underwent immunotherapy for 1.5-2 years (SIT group) and from 27 matched treated asthmatic children allergic to HDM (asthma group). After 48 h of in vitro stimulation with HDM extracts, regulatory T cells were measured by flow cytometry. Production of IL-4, IFN-gamma and TGF-beta(1 )in supernatants from allergen-stimulated cultures and the PBMC proliferations were measured by ELISA. Sera were tested for allergen-specific IgE using the ImmunoCAP 100 assay. RESULTS: Patients undergoing immunotherapy produced significantly more IL-10 and showed a significant reduction in proliferation induced by HDM extract compared with the asthma group. In cultures stimulated with HDM extract, the amounts of IL-4 and TGF-beta were lower and the amounts of IFN-gamma were higher in the SIT group compared with the asthma group. CONCLUSION: There is a functional, but quantitative, insufficiency of Treg cells in allergic asthmatic children, which was reversed in SIT-treated children. SIT can up-regulate the function of CD4(+)CD25(+)Foxp3(+) Treg cells.

Cilostazol increases tissue blood flow in contracting rabbit gastrocnemius muscle.

BACKGROUND: The mechanisms underlying the ability of cilostazol to improve walking distance in patients with intermittent claudication (IC) are not fully understood, but may be related to its phosphodiesterase type 3 (PDE3) and adenosine uptake inhibition. In the present study the effect of cilostazol on blood flow and interstitial adenosine concentration was compared with that of the PDE3 inhibitor, milrinone, and the adenosine uptake inhibitor, draflazine. METHODS AND RESULTS: Rabbit gastrocnemius muscle blood flow was measured under resting, contracting and ischemic conditions. Interstitial adenosine was sampled by microdialysis. None of the drugs affected tissue blood flow at rest. Blood flow in electrically stimulated muscle was 2- to 3-fold higher in vehicle-, milrinone- and draflazine-treated animals. However, cilostazol caused an 8-fold increase. Ligation of the femoral artery decreased blood flow in the stimulated muscle in all groups to a similar degree. Cilostazol and draflazine increased the dialysate adenosine concentration during the first 10 min of muscle contraction, but had no effect during ischemia, most likely because of the high AMP deaminase activity in skeletal muscle. CONCLUSIONS: Cilostazol increases blood flow in the gastrocnemius muscle during contraction and it is this effect that may be partially responsible for the improved walking distance in IC patients. (Circ J 2010; 74: 181 - 187).

Role of phosphodiesterase type 3A and 3B in regulating platelet and cardiac function using subtype-selective knockout mice.

Phosphodiesterase type 3 (PDE3) is an important regulator of cAMP-mediated responses within the cardiovascular system. PDE3 exists as two subtypes: PDE3A and PDE3B, with distinct cellular and subcellular locations. Due to the lack of subtype-specific pharmacological tools, the definitive role of each subtype in regulating cardiovascular function has not been determined. In this study, we investigated platelet and cardiac function, using PDE3A and PDE3B gene knockout (KO) mice. Platelet-rich-plasma was prepared from the blood of KO and age-matched wild-type (WT) mice. PGE1 (1 microg/mL) almost completely inhibited aggregation of platelets from WT, PDE3A KO and PDE3B KO mice. In platelets from WT mice, cilostamide (100 microM), a selective PDE3 inhibitor, blocked collagen- and ADP-induced aggregation. In contrast, cilostamide had no effect on aggregation of platelets from PDE3A KO mice. In PDE3B KO mice, inhibition of collagen- and ADP-induced platelet aggregation was similar to that in WT mice. The resting intra-platelet cAMP concentration in platelets from PDE3A KO mice was twice that in the WT platelets. After PGE1 (0.1 microg/mL) stimulation, intra-cellular cAMP concentration was increased significantly more in platelets from PDE3A KO mice compared to WT mice. In vivo, PDE3A KO mice were protected against collagen/epinephrine-induced pulmonary thrombosis and death, while no such protection was observed in PDE3B KO mice. The heart rate of PDE3A KO mice was significantly higher, compared with age-matched WT mice, while that of PDE3B KO mice was similar to WT. There was no difference in cardiac contractility between PDE3A or PDE3B KO mice. Heart rate and contractility were increased in a similar dose-dependent fashion by isoproterenol in both types of KO mice. Cilostamide increased heart rate and contractility in WT and PDE3B KO but not in PDE3A KO mice. Compared to WT and PDE3B KO mice, cyclic AMP-PDE activity in membrane fractions prepared from the hearts of PDE3A KO mice was lower and not inhibited by cilostamide. The data suggest that PDE3A is the main subtype of PDE3 expressed in platelets and cardiac ventricular myocytes, and is responsible for the functional changes caused by PDE3 inhibition.

The Fab fragment of a novel anti-GPVI monoclonal antibody, OM4, reduces in vivo thrombosis without bleeding risk in rats.

BACKGROUND: Inhibition of GPVI has been proposed as a useful antithrombotic strategy; however, in vivo proof-of-concept animal studies targeting GPVI are lacking. We evaluated a novel anti-human GPVI monoclonal antibody OM4 Fab in rats. METHODS AND RESULTS: OM4 Fab specifically inhibited collagen-induced aggregation of rat platelets in vitro with an IC50 of 20 to 30 microg/mL but not ADP and AA-induced platelet aggregation. After intravenous administration of OM4 Fab, a rapid inhibition of ex vivo platelet aggregation was observed with a gradual recovery within 60 to 90 minutes which corresponded to the decline in OM4 Fab plasma concentration and time-dependent decrease in platelet-bound OM4 Fab. In contrast to previous reports in mice, intravenous OM4 Fab did not deplete platelet GPVI. Injection of OM4 IgG caused acute thrombocytopenia. In a modified Folts model of cyclic flow reduction in rat carotid artery, the number of complete occlusions was significantly reduced by intravenous administration of OM4 Fab (20 mg/kg) before or after mechanical injury to the vessel, without prolongation of bleeding time. CONCLUSION: Fab fragment of the monoclonal antibody OM4 effectively inhibits collagen induced platelet aggregation in vitro and ex vivo, and in vivo thrombosis in rats without prolonging bleeding time. Antibodies against GPVI may have therapeutic potential, inhibiting thrombosis without prolonging bleeding time.