Proinflammatory and prothrombotic effects on human vascular endothelial cells of Immune-cell-derived LIGHT

Objective

LIGHT (TNFSF 14) belongs to the tumor necrosis factor superfamily and is expressed by activated T cells as well as various types of antigen presenting cells. LIGHT binds to its cellular receptors TR2 and LTßR and has a co-stimulatory role in T cell activation. Here, we compared the relative expression of LIGHT in different immune cells and the biological activity of immune cell-derived LIGHT on endothelial cells.

Methods and Results

Surface expression of LIGHT and mRNA production by PBMC and isolated T cells (CD4⁺ or CD8⁺) significantly increased after stimulation with PMA (Phorbolester-12-Myristat-13-Acetat) + ionomycin. No LIGHT expression on PMA stimulated monocytes or monocytic-like THP-1 cells could be detected; differentiation of monocytes and THP-1 cells into macrophages, however, resulted in up-regulation of LIGHT. Supernatants of stimulated T cells contained higher concentrations of soluble LIGHT than macrophage supernatants normalized to cell numbers; release of soluble LIGHT was found to be dependent on metalloproteinase activity. Size determination of released soluble LIGHT by size exclusion chromatography revealed a molecular mass of ~60 kDa, suggesting a trimeric form. Released soluble LIGHT induced expression of proinflammatory antigens ICAM-1, tissue factor and IL-8 in human endothelial cells and caused apoptosis of IFN-γ pretreated endothelial cells. Soluble LIGHT was detected at low levels in sera of healthy controls and was significantly enhanced in sera of patients with chronic hepatitis C and rheumatoid arthritis (24.93 ± 9.41 vs.129.53 ± 49.14 and 172.13 ± 77.64; p < 0.0005).

Conclusion

These findings suggest that among immune cells activated T lymphocytes are the main source of soluble LIGHT with released amounts of soluble LIGHT markedly higher compared to platelets. Immune cell-derived membrane-bound and soluble trimeric LIGHT is biologically active, inducing proinflammatory changes in endothelial cells. Enhanced plasma levels of soluble LIGHT in patients with chronic infections suggest a role of LIGHT in systemic inflammatory activation.     

Development of a theory- and evidence-based intervention to enhance implementation of physical therapy guidelines for the management of low back pain

Background

Systematic planning could improve the generally moderate effectiveness of interventions to enhance adherence to clinical practice guidelines. The aim of our study was to demonstrate how the process of Intervention Mapping was used to develop an intervention to address the lack of adherence to the national CPG for low back pain by Dutch physical therapists.

Methods

We systematically developed a program to improve adherence to the Dutch physical therapy guidelines for low back pain. Based on multi-method formative research, we formulated program and change objectives. Selected theory-based methods of change and practical applications were combined into an intervention program. Implementation and evaluation plans were developed.

Results

Formative research revealed influential determinants for physical therapists and practice quality managers. Self-regulation was appropriate because both the physical therapists and the practice managers needed to monitor current practice and make and implement plans for change. The program stimulated interaction between practice levels by emphasizing collective goal setting. It combined practical applications, such as knowledge transfer and discussion-and-feedback, based on theory-based methods, such as consciousness raising and active learning. The implementation plan incorporated the wider environment. The evaluation plan included an effect and process evaluation.

Conclusions

Intervention Mapping is a useful framework for formative data in program planning in the field of clinical guideline implementation. However, a decision aid to select determinants of guideline adherence identified in the formative research to analyse the problem may increase the efficiency of the application of the Intervention Mapping process.     

Smoking-Related Health Risks Among Persons With HIV in the Strategies for Management of Antiretroviral Therapy Clinical Trial

Objectives. We sought to determine smoking-related hazard ratios (HRs) and population-attributable risk percentage (PAR%) for serious clinical events and death among HIV-positive persons, whose smoking prevalence is higher than in the general population.Methods. For 5472 HIV-infected persons enrolled from 33 countries in the Strategies for Management of Antiretroviral Therapy clinical trial, we evaluated the relationship between baseline smoking status and development of AIDS-related or serious non-AIDS events and overall mortality.Results. Among all participants, 40.5% were current smokers and 24.8% were former smokers. Adjusted HRs were higher for current than for never smokers for overall mortality (2.4; P < .001), major cardiovascular disease (2.0; P = .002), non-AIDS cancer (1.8; P = .008), and bacterial pneumonia (2.3; P < .001). Adjusted HRs also were significantly higher for these outcomes among current than among former smokers. The PAR% for current versus former and never smokers combined was 24.3% for overall mortality, 25.3% for major cardiovascular disease, 30.6% for non-AIDS cancer, and 25.4% for bacterial pneumonia.Conclusions. Smoking contributes to substantial morbidity and mortality in this HIV-infected population. Providers should routinely integrate smoking cessation programs into HIV health care.Highly active antiretroviral therapy (HAART) for HIV has led to a decrease in AIDS-related events and deaths.^1–3 However, HIV-infected persons are also at risk for a variety of serious non–AIDS-defining diseases, including cardiovascular, renal, and hepatic disease, as well as certain cancers and infections not included in the AIDS case definition.^1,3–5 Studies conducted in the general population demonstrate that cigarette smoking increases the likelihood of many of these serious clinical conditions, including cardiovascular, pulmonary, and neoplastic diseases.^6,7 For example, smoking is a major risk factor for peripheral vascular and coronary artery disease, increasing the risk for cardiovascular disease (CVD) complications, including myocardial infarction and stroke.^6–8 Respiratory complications of smoking include chronic obstructive pulmonary disease and respiratory infections such as bacterial pneumonia or pulmonary tuberculosis.^6,7,9 Smoking increases the risk for many types of cancer, including cancers of the oral cavity, pharynx, esophagus, stomach, pancreas, larynx, lung, cervix, urinary bladder, and kidney.^6,7,10Studying smoking-related morbidity and mortality among persons with HIV is especially important because their smoking prevalence is higher than that of the general population. Twenty-one percent of US adults are current cigarette smokers,¹¹ but many recent studies have reported rates 2 or 3 times as high (46%–76%) among HIV-positive persons.^12–18 HIV-infected current smokers are reported to smoke an average of 6 to 23 cigarettes daily and to have smoked for an average of 23 to 24 years.^15,16,18,19Many smoking-related illnesses significantly affect HIV-infected persons. Lung cancer and other malignancies are important causes of death among persons with HIV.^20,21 HIV infection or use of antiretroviral drugs may contribute to CVD risk,^22,23 and use of effective HAART has resulted in increasing numbers of aging HIV-infected patients, who may develop metabolic syndrome, obesity, and other CVD risk factors.^24,25 HIV-infected patients may develop a variety of pulmonary diseases, including bacterial pneumonia^26,27; recurrent pneumonia is considered an AIDS-defining condition.²⁸Because persons with HIV are at risk for these serious and life-threatening clinical syndromes, critical prevention questions are whether and to what extent smoking further increases the risk of developing these diseases, especially in the era of HAART. Because smoking is a modifiable risk factor, it is important to define the magnitude of smoking''s effect on overall mortality among HIV-infected patients and its effect on development of specific adverse clinical conditions, including those that are not AIDS defining. It is also important to identify the proportion of disease among an HIV-infected population that is attributable to smoking. This information can be used to help estimate the effect of smoking cessation on reducing disease and improving survival for HIV-infected persons and to counsel individual patients about ways to optimize their health.We evaluated data from a large multisite international study of 2 HAART treatment strategies. We determined the relative risks associated with smoking for development of different serious clinical events and on all-cause mortality, with adjustment for a variety of important potential confounders. We also calculated the population-attributable risk percentage (PAR%) associated with smoking for these clinical syndromes and all-cause mortality.     

Molecular requirements for inhibition of the chemokine receptor CCR8 – probe-dependent allosteric interactions

BACKGROUND AND PURPOSE

Here we present a novel series of CCR8 antagonists based on a naphthalene-sulfonamide structure. This structure differs from the predominant pharmacophore for most small-molecule CC-chemokine receptor antagonists, which in fact activate CCR8, suggesting that CCR8 inhibition requires alternative structural probes.

EXPERIMENTAL APPROACH

The compounds were tested as inverse agonists and as antagonists against CCL1-induced activity in Gα_i signalling and chemotaxis. Furthermore, they were assessed by heterologous competition binding against two radiolabelled receptor ligands: the endogenous agonist CCL1 and the virus-encoded antagonist MC148.

KEY RESULTS

All compounds were highly potent inverse agonists with EC₅₀ values from 1.7 to 23 nM. Their potencies as antagonists were more widely spread (EC₅₀ values from 5.9 to 1572 nM). Some compounds were balanced antagonists/inverse agonists whereas others were predominantly inverse agonists with >100-fold lower potency as antagonists. A correspondingly broad range of affinities, which followed the antagonist potencies, was disclosed by competition with [¹²⁵I]-CCL1 (K_i 3.4–842 nM), whereas the affinities measured against [¹²⁵I]-MC148 were less widely spread (K_i 0.37–27 nM), and matched the inverse agonist potencies.

CONCLUSION AND IMPLICATIONS

Despite highly potent and direct effects as inverse agonists, competition-binding experiments against radiolabelled agonist and tests for antagonism revealed a probe-dependent allosteric effect of these compounds. Thus, minor chemical changes affected the ability to modify chemokine binding and action, and divided the compounds into two groups: predominantly inverse agonists and balanced antagonists/inverse agonists. These studies have important implications for the design of new inverse agonists with or without antagonist properties.     

Blood-brain barrier in vitro models as tools in drug discovery: assessment of the transport ranking of antihistaminic drugs

In the course of our validation program testing blood-brain barrier (BBB) in vitro models for their usability as tools in drug discovery it was evaluated whether an established Transwell model based on porcine cell line PBMEC/C1-2 was able to differentiate between the transport properties of first and second generation antihistaminic drugs. First generation antihistamines can permeate the BBB and act in the central nervous system (CNS), whereas entry to the CNS of second generation antihistamines is restricted by efflux pumps such as P-glycoprotein (P-gP) located in brain endothelial cells. P-gP functionality of PBMEC/C1-2 cells grown on Transwell filter inserts was proven by transport studies with P-gP substrate rhodamine 123 and P-gP blocker verapamil. Subsequent drug transport studies with the first generation antihistamines promethazine, diphenhydramine and pheniramine and the second generation antihistamines astemizole, ceterizine, fexofenadine and loratadine were accomplished in single substance as well as in group studies. Results were normalised to diazepam, an internal standard for the transcellular transport route. Moreover, effects after addition of P-gP inhibitor verapamil were investigated. First generation antihistamine pheniramine permeated as fastest followed by diphenhydramine, diazepam, promethazine and second generation antihistaminic drugs ceterizine, fexofenadine, astemizole and loratadine reflecting the BBB in vivo permeability ranking well. Verapamil increased the transport rates of all second generation antihistamines, which suggested involvement of P-gP during their permeation across the BBB model. The ranking after addition of verapamil was significantly changed, only fexofenadine and ceterizine penetrated slower than internal standard diazepam in the presence of verapamil. In summary, permeability data showed that the BBB model based on porcine cell line PBMEC/C1-2 was able to reflect the BBB in vivo situation for the transport of antihistaminc drugs and to distinguish between first and second generation antihistamines.     

Seroprevalence of Strongyloides stercoralis in a South Australian Vietnam veteran cohort

Objectives: Strongyloides stercoralis is a parasitic roundworm causing chronic infection that is endemic in Southeast Asia. Vietnam veterans are considered to be at high risk of Strongyloides infection. The prevalence of persistent infection in this group is unknown. This study aimed to establish the seropositivity rate of Strongyloides antibodies in South Australian Vietnam veterans and to identify the most reported symptoms within the seropositive group. Method: This cross‐sectional study recruited 309 veterans who had served in Vietnamese territory between 1962 and 1975 and were currently resident in South Australia. Participants completed a self‐administered questionnaire examining demographics, deployment, somatic symptoms and depression. Venous blood was collected for Strongyloides serology and eosinophil count. Participants who demonstrated positive Strongyloides serology underwent faecal microscopy for parasites. Results: A total of 309 participants were recruited and 256 completed the questionnaire. Strongyloides seropositivity was demonstrated in 29 of 249 participants (11.6%). No participant had Strongyloides larvae detected by faecal microscopy. On multivariate analysis, only dermatological symptoms were associated with positive serology (OR 4.84, 95%CI 1.31–17.92, p‐value 0.01). Conclusions: This study found a high seroprevalence (11.6%) of Strongyloides antibodies within the Vietnam veteran community in South Australia. Seropositivity was associated with increased likelihood of dermatological symptoms. Implications: Post‐deployment screening and eradication therapy for Strongyloides should be offered to ADF staff after service in Strongyloides‐endemic areas. This should include those veterans who left the service many years ago.     

Method of evaluating and improving ambulatory medical care.

The usefulness of an action-research model is demonstrated in the evaluation and improvement of ambulatory medical care in a variety of settings: solo office practice, prepaid capitation multiple-specialty group practice, and medical school hospital-based outpatient clinic practice. Improvements in the process of medical care are found to relate directly to the intensity and duration of planned interventions by the study group and are demonstrated to follow organizational changes in the participating sites--primarily managerial and support services initiated by policy decisions in each study site. Improvement in performance approaching one standard deviation results from the most intense intervention, about one-half standard deviation at the next level of intervention, and virtually no change from a simple feedback of performance measures. On the basis of these findings and other operational and research efforts to improve physician performance, it is unlikely that simple feedback of performance measures will elicit a change in behavior. However, noncoercive methods involving health care providers in problem identification, problem solving, and solution implementation are demonstrated to be effective.     

Quadruple tacrolimus-based induction therapy including azathioprine and ALG does not significantly improve outcome after liver transplantation when compared with standard induction with tacrolimus and steroids: results of a prospective, randomized trial

BACKGROUND: Tacrolimus in combination with prednisolone has been proven to be a safe and effective immunosuppressive induction therapy in solid organ transplantation. However, it remains unclear whether a tacrolimus-based quadruple induction regimen with azathioprine and an antilymphocytic preparation could further improve the results after orthotopic liver transplantation. Therefore, we designed a prospective, randomized study to compare the immunosuppressive efficacy of dual (tacrolimus and prednisolone) and quadruple (tacrolimus, azathioprine, ALG Merieux and prednisolone) induction after liver transplantation. METHODS: After randomization, 120 consecutive patients of primary liver transplants were divided into the dual group (n=59) and the quadruple group (n=61) and followed for a minimum of 3 years. RESULTS: Patient survival at 3 years was 88.2% in the dual versus 94.9% in the quadruple group. Overall 25 patients in each group (41 and 42%, respectively) developed acute rejection. There was no difference in the number and severity of rejections. In each group only four patients required OKT3-therapy, however, although three of four patients in the quadruple group responded to OKT3 and cleared rejection, none of the four patients in the dual group were treated successfully with OKT3 (P<0.02). Rejection in these patients resolved only after additional treatment with mycophenolate mofetil. Adverse events and infections were equally distributed in both groups. Asymptomatic Cytomegalovirus infections were more common in the quadruple group (P<0.02). As of today, only one patient developed posttransplant lymphoproliferative disease (dual group). CONCLUSIONS: The data from our single-center study indicate that both tacrolimus-based dual and quadruple immunosuppressive induction regimens yield similar safety and effectiveness after liver transplantation.     

Influence of Gases on Intraperitoneal Immunity during Laparoscopy in Tumor-bearing Rats

Laparoscopy has been associated with metastases to abdominal wall wounds. In addition, many recent experimental studies suggest that laparoscopy is associated with increased tumor dissemination. It is possible that immune or metabolic disturbances due to the use of a pneumoperitoneum could contribute to this problem. To investigate this possibility, we studied the effect of two insufflation gases and gasless laparoscopy on in vivo peritoneal macrophage function and intraperitoneal pH in an experimental model. A carcinoma was implanted into the flank of 32 experimental rats that underwent laparoscopic surgery in one of four treatment groups: anesthesia alone, gasless laparoscopy, helium insufflation, and CO₂ insufflation. Intraperitoneal pH was monitored during surgery, and peritoneal macrophage function was determined 3 days after surgery by harvesting peritoneal macrophages and then examining their ability to produce tumour necrosis factor-α (TNF-α). CO₂ insufflation was associated with a consistent fall in intraperitoneal pH and a significant reduction in TNFα production. These findings did not occur in the other study groups. The results of this study demonstrate that CO₂ insufflation results in depressed intraperitoneal macrophage activity. It is possible that it is mediated by pH changes. In addition, it could be a contributing factor to the development of port-site metastases. Further studies are needed to determine whether the factors identified act during clinical surgery.     

Follow-up of patients with anterior wall aneurysms using radionuclide ventriculography

32 patients with a large anterolateral aneurysm were studied by using biplane ventricular angiography and rest and exercise radionuclide ventriculography (gated-blood-pool method). The correlation coefficient of biplane ejection fraction by angiography and radionuclide ventriculography was poor (r = 0.65), but repeated investigations and interobserver comparison showed nearly identical values, therefore we used the gated-blood-pool method (GBP) for follow-up investigations in patients with an aneurysm of the anterolateral wall. 11 patients were studied before and 17 +/- 6 months after left ventricular aneurysmectomy. Resting ejection fraction (LVEF) increased significantly from 28 +/- 8 to 38 +/- 8% (p less than 0.005), but exercise LVEF did not. In 21 patients with medical therapy the second GBP measurement after 16 +/- 6 months showed so significant changes in biplane LVEF (33 +/- 9 and 31 +/- 7%, respectively). During exercise LVEF remained unchanged after aneurysmectomy but increased slightly by 5% (p less than 0.05) in the medically treated group. The regional wall motion was unchanged in patients with medical therapy, but after aneurysmectomy there was a significant increase in local ejection fractions in the anterolateral, apical and posterolateral region. The gated-blood-pool methods is suitable for the determination of left ventricular function in patients with anterolateral aneurysm and may be used for follow-up studies. Left ventricular aneurysmectomy is effective in improving resting ventricular function, whereas in patients with medical therapy LV function remains unchanged.