Imbalance between subsets of CD8(+) peripheral blood T cells in patients with chronic obstructive pulmonary disease.

Background. CD8(+) T lymphocytes are known to play a critical role in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, systematic analyses of CD8(+) T cell (Cytotoxic T cells, Tc) subsets in COPD patients have yet to be well conducted. Methods. The whole Tc subsets, including Tc1/2/10/17, CD8(+) regulatory T cells (Tregs) and CD8(+) α7(+) T cells, were quantified by flow cytometry in peripheral blood from 24 stable COPD subjects (SCOPD), 14 patients during acute exacerbations (AECOPD), and 14 healthy nonsmokers (HN). Results. Acute exacerbations of COPD were accompanied by elevated levels of circulating CD8(+) T cells. Tc1 cells were increased in both SCOPD and AECOPD patients, whereas the percentage of Tc2 cells was decreased in SCOPD patients but remained normal in AECOPD patients. Tc17 cells were increased only in AECOPD patients, and the percentage of Tc10 cells was reduced in both SCOPD and AECOPD patients. The imbalances of pro/anti-inflammatory Tc subsets observed in COPD may be caused by the lack of Tc10 cells and the impaired anti-inflammatory capacity of CD8(+) Tregs. Conclusions. The imbalances between subsets of CD8(+) peripheral blood T cells contribute to the immune response dysfunction in COPD pathogenesis.

Efficacy and Safety of Tiotropium in the Treatment of Severe Persistent Asthma:Meta-analysis.

OBJECTIVE: To evaluate the efficacy and safety of tiotropium in treatment of severe persistent asthma. METHODS: Reports of randomized controlled trials (RCTs) describing tiotropium for treatment of severe persistent asthma published from January 1946 to February 2015 were searched in Cochrane Library, ClinicalTrials.gov, PubMed, Ovid Medline, CNKI, and CSJD. The data of the included RCTs were extracted and the data quality was evaluated. Meta-analyses were performed with Revman 5.3 software. RESULTS: Five RCTs including 1433 patients were analyzed. Meta-analysis of the data showed that compared with the placebo group, tiotropium treatment significantly improved the patients' peak forced expiratory volume in one second (FEV1) [weighted mean difference (WMD): 0.13 L, 95% confidence interval (CI): 0.10-0.16 L, P<0.00001], trough FEV1 (WMD: 0.09 L, 95%CI: 0.06-0.12 L, P<0.00001), peak forced vital capacity (FVC) (WMD: 0.10 L, 95%CI: 0.06-0.14 L, P<0.00001), trough FVC (WMD: 0.12 L, 95%CI: 0.08-0.17 L, P<0.00001), morning peak expiratory flow (PEF) (WMD: 9.21 L/min, 95%CI: 4.2-14.23 L/min, P=0.0003), evening PEF (WMD: 22.06 L/min, 95%CI 13.05-31.08 L/min, P<0.00001). The scores of asthma control questionnaire (ACQ) (WMD: 0.01, 95% CI: -0.07-0.09, P=0.86) or asthma quality of life questionnaire (AQLQ)(WMD: 0.06, 95% CI:-0.18-0.06, P=0.33) were not affected by tiotropium. No significant difference with adverse events between tiotropium group and placebo group were reported in these included studies (P>0.05). CONCLUSIONS: Tiotropium for severe persistent asthma treatment can improve FEV1, FVC, and PEF but may not improve the quality of life of the patients. Tiotropium is well tolerated and can be an add-on therapy for severe persistent asthma.

[Clinical Analysis of 35 Inpatients with Diffuse Panbronchiolitis in Peking Union Medical College Hospital].

OBJECTIVE: To summarize the clinical features of diffuse panbronchiolitis (DPB). METHOD: We retrospectively analyzed the clinical data of 35 patients who had been admitted to Peking Union Medical College Hospital from December 1996 to July 2014 due to DPB,which was confirmed basing on the diagnostic criteria proposed in 1998 by a working group of the Ministry of Health and Welfare of Japan or histopathological examination. RESULTS: The average age of these 35 patients (20 men and 15 women,with a sex ratio of 1.33 to 1) was (42.2<15.6) years,mainly distributed in the 40-49 age group. The average clinical history was (8.4<8.5) years. The main symptoms and signs of DPB included chronic cough (n=35,100%),copious purulent sputum production (n=31,88.6%),exertional dyspnoea (n=24,68.6%),end-inspiratory crackles (n=28,80.0%). Also,26 patients (74.3%) had a history of sinusitis. Cold agglutination test in 15 out of 15 patients were negative. Pseudomonas aeruginosa and Haemophilus influenza were isolated from 22 patients (73.3%,22/30),and 26 patients (83.9%,26/31) had hypoxemia. The mean values of forced expiratory volume in the first second/forced vital capacity,residua volume/total lung volume,maximum forced expiratory volume of 50% lung volume,and maximum forced expiratory volume of 25% lung volume were 60.5%,53.8%,25.9%,and 31.2%,respectively. The most common CT findings from this cohort of patients were bronchiectasis and bronchiolitis,with nodular shadows distributed in a centrilobular pattern. Finally,29 patients were misdiagnosed as other conditions such as pulmonary infection and bronchiectasis. CONCLUSIONS: DPB in Chinese populations have different presentations compared to that Japanese populations:for instance,the serum cold agglutination test always shows negative results,which is often inconsistent with the pathogens in sputum. DPB usually is misdiagnosed. Clinicians should take DPB into consideration when patients had pulmonary infection and sinusitis.

Cigarette smoking promotes inflammation in patients with COPD by affecting the polarization and survival of Th/Tregs through up-regulation of muscarinic receptor 3 and 5 expression.

BACKGROUND: CD4+ T cells in the lung are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD), although CD4+ T cell subsets and the direct effect of smoking on these cells, especially the expression of MRs, have not been comprehensively examined. METHODS: First, circulating CD4+ T cell subsets in healthy nonsmokers, patients with SCOPD and patients with AECOPD were evaluated by flow cytometry. Then, differentiation experiments were carried out using RT-PCR, and Ki-67/Annexin V antibodies were used to measure proliferation and apoptosis. We also explored the impact of CSE on the differentiation and survival of CD4+Th/Tregs and examined the expression of MRs in healthy nonsmokers and patients with SCOPD. RESULTS: We found the percentages of circulating Th1 and Th17 cells were increased in patients with AECOPD, while the percentage of Th2 cells was decreased in patients with SCOPD. The percentages of Th10 cells were decreased in both patients with SCOPD and patients with AECOPD, while the percentages of Tregs were increased. In addition, the percentages of CD4+α-7+ T cells were decreased in patients with SCOPD and patients with AECOPD. However, only the decrease observed in patients with AECOPD was significant. In vitro studies also revealed MR expression affected the polarization of T cells, with different CD4+ T cell subtypes acquiring different MR expression profiles. The addition of CSE facilitated CD4+ T cell polarization towards pro-inflammatory subsets (Th1 and Th17) and affected the survival of CD4+ T cells and Treg cells by up-regulating the expression of MR3 and 5, resulting in an imbalance of CD4+ T cell subsets. CONCLUSIONS: Our findings suggest an imbalance of circulating CD4+ T cell subsets is involved in COPD pathogenesis in smokers. Cigarette smoking may contribute to this imbalance by affecting the polarization and survival of Th/Tregs through the up-regulation of MR3 and MR5.

Treg/IL-17 ratio and Treg differentiation in patients with COPD.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by chronic pulmonary and systematic inflammation. An abnormal adaptive immune response leads to an imbalance between pro- and anti-inflammatory processes. T-helper (Th), T-cytotoxic (Tc) and T-regulatory (Treg) cells may play important roles in immune and inflammatory responses. This study was conducted to clarify the changes and imbalance of cytokines and T lymphocyte subsets in patients with COPD, especially during acute exacerbations (AECOPD). METHODS: Twenty-three patients with stable COPD (SCOPD) and 21 patients with AECOPD were enrolled in the present study. In addition, 20 age-, sex- and weight-matched non-smoking healthy volunteers were included as controls. The serum levels of selected cytokines (TGF-ß, IL-10, TNF-α, IL-17 and IL-9) were measured by enzyme-linked immunosorbent assay (ELISA) kits. Furthermore, the T lymphocyte subsets collected from peripheral blood samples were evaluated by flow cytometry after staining with anti-CD3-APC, anti-CD4-PerCP, anti-CD8- PerCP, anti-CD25-FITC and anti-FoxP3-PE monoclonal antibodies. Importantly, to remove the confounding effects of inflammatory factors, the authors introduced a concept of "inflammation adjustment" and corrected each measured value using representative inflammatory markers, such as TNF-α and IL-17. RESULTS: Unlike the other cytokines, serum TGF-ß levels were considerably higher in patients with AECOPD relative to the control group regardless of adjustment. There were no significant differences in the percentages of either CD4+ or CD8+ T cells among the three groups. Although Tregs were relatively upregulated during acute exacerbations, their capacities of generation and differentiation were far from sufficient. Finally, the authors noted that the ratios of Treg/IL-17 were similar among groups. CONCLUSIONS: These observations suggest that in patients with COPD, especially during acute exacerbations, both pro-inflammatory and anti-inflammatory reactions are strengthened, with the pro-inflammatory reactions dominating. Although the Treg/IL-17 ratios were normal, the regulatory T cells were still insufficient to suppress the accompanying increases in inflammation. All of these changes suggest a complicated mechanism of pro- and anti-inflammatory imbalance which needs to be further investigated.

Dexmedetomidine alleviates anxiety-like behaviors and cognitive impairments in a rat model of post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a psychiatric disease that has substantial health implications, including high rates of health morbidity and mortality, as well as increased health-related costs. Although many pharmacological agents have proven the effects on the development of PTSD, current pharmacotherapies typically only produce partial improvement of PTSD symptoms. Dexmedetomidine is a selective, short-acting α2-adrenoceptor agonist, which has anxiolytic, sedative, and analgesic effects. We therefore hypothesized that dexmedetomidine possesses the ability to prevent the development of PTSD and alleviate its symptoms. By using the rat model of PTSD induced by five electric foot shocks followed by three weekly exposures to situational reminders, we showed that the stressed rats displayed pronounced anxiety-like behaviors and cognitive impairments compared to the controls. Notably, repeated administration of 20µg/kg dexmedetomidine showed impaired fear conditioning memory, decreased anxiety-like behaviors, and improved spatial cognitive impairments compared to the vehicle-treated stressed rats. These data suggest that dexmedetomidine may exert preventive and protective effects against anxiety-like behaviors and cognitive impairments in the rats with PTSD after repeated administration.

Flurbiprofen improves dysfunction of T-lymphocyte subsets and natural killer cells in cancer patients receiving post-operative morphine analgesia.

OBJECTIVE: Acute pain can lead to immune dysfunction, which can be partly ameliorated by successful pain management. Opioids, which are widely used for analgesia, can result in the deterioration of immune function. This study aimed to investigate the influence of morphine with or without flurbiprofen as post-operative analgesics on the immune systems of patients undergoing gastric cancer surgery. METHODS: 60 patients undergoing gastric cancer surgery were equally randomized into two groups. They received post-operative patient-controlled intravenous (IV) analgesia using morphine either with or without flurbiprofen. Visual analogue scale (VAS) scores, Bruggemann comfort scale (BCS) scores, morphine consumption, time of first flatus, incidence of nausea/vomiting, and T-lymphocyte subsets (CD3⁺, CD4⁺, and CD8⁺) and natural killer cells (CD3⁻CD16⁺CD56⁺) were evaluated. RESULTS: No significant difference was observed in the VAS scores, BCS scores, and nausea/vomiting incidence between groups. Less morphine was consumed and the time of first flatus was earlier in patients receiving morphine with flurbiprofen than morphine alone. The expression of CD3⁺, CD4⁺, CD4⁺/CD8⁺, and CD3⁻CD16⁺CD56⁺ decreased at 2 hours after incision and, except for CD3⁻CD16⁺CD56⁺, returned to baseline at 120 hours after surgery. Moreover, the expression of CD3⁻CD16⁺CD56⁺ at 2 hours after incision and the expression of CD3⁺, CD4⁺, CD4⁺/CD8⁺, and CD3⁻CD16⁺CD56⁺ at 24 hours after surgery were higher in patients receiving morphine with flurbiprofen than morphine alone. CONCLUSION: The combination of morphine and flurbiprofen ameliorates the immune depression in Tlymphocyte subsets and natural killer cells and provides a similar analgesic efficacy to morphine alone in patients undergoing gastric cancer surgery.

Recombinant strains for the enhanced production of bioengineered rapalogs.

The rapK gene required for biosynthesis of the DHCHC starter acid that initiates rapamycin biosynthesis was deleted from strain BIOT-3410, a derivative of Streptomyces rapamycinicus which had been subjected to classical strain and process development and capable of robust rapamycin production at titres up to 250mg/L. The resulting strain BIOT-4010 could no longer produce rapamycin, but when supplied exogenously with DHCHC produced rapamycin at titres equivalent to its parent strain. This strain enabled mutasynthetic access to new rapalogs that could not readily be isolated from lower titre strains when fed DHCHC analogs. Mutasynthesis of some rapalogs resulted predominantly in compounds lacking late post polyketide synthase biosynthetic modifications. To enhance the relative production of fully elaborated rapalogs, genes encoding late-acting biosynthetic pathway enzymes which failed to act efficiently on the novel compounds were expressed ectopically to give strain BIOT-4110. Strains BIOT-4010 and BIOT-4110 represent valuable tools for natural product lead optimization using biosynthetic medicinal chemistry and for the production of rapalogs for pre-clinical and early stage clinical trials.

Working with small molecules: rules-of-thumb of "drug likeness".

Based on analyses of existing small organic drug molecules, a set of "rules-of-thumb" have been devised to assess the likeness of a small molecule under study to those existing drugs in terms of physicochemical and topological properties. These rules can be used to estimate the likelihood of a small molecule to possess the desired efficacy, pharmacokinetic/pharmacodynamic properties, and toxicity profiles to eventually become a drug, and therefore, whether it justifies further experimental work and development. These rules are particularly useful when selecting a chemical starting point for a given project or choosing a chemical series to focus when multiple series are available. Caution should be paid, however, not to overly rely on these rules for decision-making, since these rules are restricted by knowledge of existing drugs. Novel chemotypes and/or targets may be exceptions.

Preclinical characterization of naturally occurring polyketide cyclophilin inhibitors from the sanglifehrin family.

Cyclophilin inhibitors currently in clinical trials for hepatitis C virus (HCV) are all analogues of cyclosporine (CsA). Sanglifehrins are a group of naturally occurring cyclophilin binding polyketides that are structurally distinct from the cyclosporines and are produced by a microorganism amenable to biosynthetic engineering for lead optimization and large-scale production by fermentation. Preclinical characterization of the potential utility of this class of compounds for the treatment of HCV revealed that the natural sanglifehrins A to D are all more potent than CsA at disrupting formation of the NS5A-CypA, -CypB, and -CypD complexes and at inhibition of CypA, CypB, and CypD isomerase activity. In particular, sanglifehrin B (SfB) was 30- to 50-fold more potent at inhibiting the isomerase activity of all Cyps tested than CsA and was also shown to be a more potent inhibitor of the 1b subgenomic replicon (50% effective concentrations [EC50s] of 0.070 µM and 0.16 µM in Huh 5-2 and Huh 9-13 cells, respectively). Physicochemical and mouse pharmacokinetic analyses revealed low oral bioavailability (F<4%) and low solubility (<25 µM), although the half-lives (t1/2) of SfA and SfB in mouse blood after intravenous (i.v.) dosing were long (t1/2>5 h). These data demonstrate that naturally occurring sanglifehrins are suitable lead compounds for the development of novel analogues that are less immunosuppressive and that have improved metabolism and pharmacokinetic properties.

Region of interest tracking in real-time myocardial contrast echocardiography.

The real-time myocardial contrast echocardiography (RT MCE) is a new echocardiography technology, which allows clinicians to noninvasively evaluate the perfusion of myocardial capillary of patients, using the quantitative analysis of RT MCE. But the accurate analysis requires tracking the position of region of interest (ROI) within the myocardial area, so as to compensate for the translation or rotation offsets, which are due to such uncontrollable factors as heart motion. We used diamond search method and Brox's coarse-to-fine warping optical flow technique for this ROI tracking problem. We validated our methods by comparing the quantitative analysis results of RT MCE using our methods with those using Lucas & Kanade's optical flow technique, which had been report to be accurate enough for this ROI tracking. We finally present some examples of animal experiment to show the effectiveness and the clinical application value of our ROI tracking methods.

Numerical methods and workstation for the quantitative analysis of real-time myocardial contrast echocardiography.

Using the quantitative analysis of real-time myocardial contrast echocardiography (RT MCE), clinicians can assess the myocardial perfusion of patients, noninvasively and accurately. We designed a workstation to assist clinicians to automatically implement the accurate analysis of RT MCE. The workstation can compute some hemodynamic parameters of myocardial microcirculation, e.g., myocardial blood flow, myocardial blood flow mean velocity, and myocardial blood volume. Our new methods involved in the quantitative analysis of RT MCE are summarized as follows. 1) A novel orthogonal array optimization (OAO) technique was proposed and used to estimate the unknown parameters of the nonlinear model to guarantee numerical stability. 2) Brox's coarse-to-fine warping optical flow technique was employed to automatically track the region of interest located inside the myocardial area to ensure the accuracy of the quantitative analysis. Finally, we illustrate some examples of clinical studies to indicate the effectiveness of the system and the reliability of the methods.

Optimizing natural products by biosynthetic engineering: discovery of nonquinone Hsp90 inhibitors.

A biosynthetic medicinal chemistry approach was applied to the optimization of the natural product Hsp90 inhibitor macbecin. By genetic engineering, mutants have been created to produce novel macbecin analogues including a nonquinone compound (5) that has significantly improved binding affinity to Hsp90 (Kd 3 nM vs 240 nM for macbecin) and reduced toxicity (MTD > or = 250 mg/kg). Structural flexibility may contribute to the preorganization of 5 to exist in solution in the Hsp90-bound conformation.

Molecular characterization of macbecin as an Hsp90 inhibitor.

Macbecin compares favorably to geldanamycin as an Hsp90 inhibitor, being more soluble, stable, more potently inhibiting ATPase activity (IC50 = 2 microM) and binding with higher affinity (Kd = 0.24 microM). Structural studies reveal significant differences in their Hsp90 binding characteristics, and macbecin-induced tumor cell growth inhibition is accompanied by characteristic degradation of Hsp90 client proteins. Macbecin significantly reduced tumor growth rates (minimum T/C: 32%) in a DU145 murine xenograft. Macbecin thus represents an attractive lead for further optimization.

Drug discovery beyond the 'rule-of-five'.

Although a very useful guideline for orally bioavailable small-molecule drug design, the 'rule-of-five' (also known as 'Lipinski's rule of drug-likeness') has to some extent been overemphasized. Firstly, only 51% of all FDA-approved small-molecule drugs are both used orally and comply with the 'rule-of-five'. This does not even include the increasing number of biologicals of which several have reached 'blockbuster' status. Secondly, it does not cover natural product and semisynthetic natural product drugs, which constitute over one-third of all marketed small-molecule drugs. A more balanced and programmatic approach to drug discovery should be more productive than to rely on an overemphasis of 'rule-of-five' compliance. Rather it should consider proactively the development of parenteral drugs in parallel to oral drugs and to consider the development of therapeutic antibodies in parallel to small-molecule drugs. These are particularly relevant for efforts against 'first-in-class' and/or particularly challenging targets such as proteases and those involving protein-protein interactions. In addition, more effort should be invested in natural product research. Emerging novel technologies such as synthetic biology (genetic engineering of living organisms to produce small-molecule therapeutics) may address several challenging issues of natural product-based drug discovery including synthetic feasibility and ligand efficiency.

Separation of anti-angiogenic and cytotoxic activities of borrelidin by modification at the C17 side chain.

A set of novel borrelidin analogues have been prepared by precursor-directed biosynthesis. Structure-activity relationship analysis suggests that steric structural arrangement within the C17 side chain is important for differentiating cytotoxic and anti-angiogenic activities. A C17-cyclobutyl analogue 3 was found to have markedly increased selectivity for in vitro angiogenesis inhibition over cytotoxicity and is therefore potentially useful as an anticancer agent.

Biosynthesis of the angiogenesis inhibitor borrelidin: directed biosynthesis of novel analogues.

We report the directed biosynthesis of borrelidin analogues and their selective anti-proliferative activity against human cancer cell lines.