Lipoxins modulate neutrophil oxidative burst, integrin expression and lymphatic transmigration differentially in human health and atherosclerosis.

Dysregulated chronic inflammation plays a crucial role in the pathophysiology of atherosclerosis and may be a result of impaired resolution. Thus, restoring levels of specialized pro-resolving mediators (SPMs) to promote the resolution of inflammation has been proposed as a therapeutic strategy for patients with atherosclerosis, in addition to standard clinical care. Herein, we evaluated the effects of the SPM lipids, lipoxin A4 (LXA4 ) and lipoxin B4 (LXB4 ), on neutrophils isolated from patients with atherosclerosis compared with healthy controls. Patients displayed altered endogenous SPM production, and we demonstrated that lipoxin treatment in whole blood from atherosclerosis patients attenuates neutrophil oxidative burst, a key contributor to atherosclerotic development. We found the opposite effect in neutrophils from healthy controls, indicating a potential mechanism whereby lipoxins aid the endogenous neutrophil function in health but reduce its excessive activation in disease. We also demonstrated that lipoxins attenuated upregulation of the high-affinity conformation of the CD11b/CD18 integrin, which plays a central role in clot activation and atherosclerosis. Finally, LXB4 enhanced lymphatic transmigration of human neutrophils isolated from patients with atherosclerosis. This finding is noteworthy, as impaired lymphatic function is now recognized as an important contributor to atherosclerosis. Although both lipoxins modulated neutrophil function, LXB4 displayed more potent effects than LXA4 in humans. This study highlights the therapeutic potential of lipoxins in atherosclerotic disease and demonstrates that the effect of these SPMs may be specifically tailored to the need of the individual.

Annexin A1 in blood mononuclear cells from patients with coronary artery disease: Its association with inflammatory status and glucocorticoid sensitivity.

Annexin A1 (AnxA1) is a key player in resolution of inflammation and a mediator of glucocorticoid actions. In atherosclerotic tissue, increased expression of AnxA1 has been associated with protective plaque-stabilizing effects. Here, we investigated the expression of AnxA1 in peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease (CAD). Blood was collected from 57 patients with stable CAD (SCAD) and 41 healthy controls. We also included a minor group (n = 10) with acute coronary syndrome (ACS). AnxA1 mRNA was measured in PBMCs. Expression of AnxA1 protein (total and surface-bound) and glucocorticoid receptors (GR) were detected in PBMC subsets by flow cytometry. Also, salivary cortisol, interleukin(IL)-6 and IL-10 in plasma, and LPS-induced cytokine secretion from PBMCs, with or without dexamethasone, were assessed. AnxA1 mRNA was found to be slightly increased in PBMCs from SCAD patients compared with controls. However, protein expression of AnxA1 or GRs in PBMC subsets did not differ between SCAD patients and controls, despite SCAD patients showing a more proinflammatory cytokine profile ex vivo. Only surface expression of AnxA1 on monocytes correlated with dexamethasone-mediated suppression of cytokines. In ACS patients, a marked activation of AnxA1 was seen involving both gene expression and translocation of protein to cell surface probably reflecting a rapid glucocorticoid action modulating the acute inflammatory response in ACS. To conclude, surface expression of AnxA1 on monocytes may reflect the degree of glucocorticoid sensitivity. Speculatively, "normal" surface expression of AnxA1 indicates that anti-inflammatory capacity is impaired in SCAD patients.

Persistent accumulation of interferon-γ-producing CD8+CD56+ T cells in blood from patients with coronary artery disease.

OBJECTIVE: There is emerging evidence for CD8(+) T cell alterations in blood from patients with coronary artery disease (CAD). We examined whether the distribution and phenotype of CD8(+)CD56(+) T cells differed according to the clinical manifestation of CAD. METHODS: Patients with acute coronary syndrome (ACS, n = 30), stable angina (SA, n = 34) and controls (n = 36) were included. Blood was collected before and up to 12 months after referral for coronary investigation. CD8(+)CD56(+) T cells were assessed by flow cytometry for expression of surface markers, apoptosis, and intracellular expression of cytokines. RESULTS: The proportions of CD8(+)CD56(+) T cells were significantly higher in both ACS and SA patients compared with controls, and remained so after 3 and 12 months. This was independent of age, sex, systemic inflammation and cytomegalovirus seropositivity. CD8(+)CD56(+) T cells differed from CD8(+)CD56(-) T cells in terms of lower CD28 expression and fewer apoptotic cells. Both CD8(+) T cell subsets were positive for interferon (IFN)-γ and tumor necrosis factor, although IFN-γ was significantly more confined to the CD8(+)CD56(+) T cells. CONCLUSION: The persistent accumulation of CD8(+)CD56(+) T cells in ACS and SA patients share several features with immunological aging. It also contributes to a larger IFN-γ(+) pool in blood, and may thereby hypothetically drive the atherosclerotic process in a less favorable direction.

Common genetic variations in the NALP3 inflammasome are associated with delayed apoptosis of human neutrophils.

BACKGROUND: Neutrophils are key-players in the innate host defense and their programmed cell death and removal are essential for efficient resolution of inflammation. These cells recognize a variety of pathogens, and the NOD-like receptors (NLRs) have been suggested as intracellular sensors of microbial components and cell injury/stress. Some NLR will upon activation form multi-protein complexes termed inflammasomes that result in IL-1ß production. NLR mutations are associated with auto-inflammatory syndromes, and our previous data propose NLRP3 (Q705K)/CARD-8 (C10X) polymorphisms to contribute to increased risk and severity of inflammatory disease by acting as genetic susceptibility factors. These gene products are components of the NALP3 inflammasome, and approximately 6.5% of the Swedish population are heterozygote carriers of these combined gene variants. Since patients carrying the Q705K/C10X polymorphisms display leukocytosis, the aim of the present study was to find out whether the inflammatory phenotype was related to dysfunctional apoptosis and impaired clearance of neutrophils by macrophages. METHODS AND FINDINGS: Patients carrying the Q705K/C10X polymorphisms displayed significantly delayed spontaneous as well as microbe-induced apoptosis compared to matched controls. Western blotting revealed increased levels and phosphorylation of Akt and Mcl-1 in the patients' neutrophils. In contrast to macrophages from healthy controls, macrophages from the patients produced lower amounts of TNF; suggesting impaired macrophage clearance response. CONCLUSIONS: The Q705K/C10X polymorphisms are associated with delayed apoptosis of neutrophils. These findings are explained by altered involvement of different regulators of apoptosis, resulting in an anti-apoptotic profile. Moreover, the macrophage response to ingestion of microbe-induced apoptotic neutrophils is altered in the patients. Taken together, the patients display impaired turnover and clearance of apoptotic neutrophils, pointing towards a dysregulated innate immune response that influences the resolution of inflammation. The future challenge is to understand how microbes affect the activation of inflammasomes, and why this interaction will develop into severe inflammatory disease in certain individuals.

Different perspectives on communication quality and emotional functioning during routine oncology consultations.

OBJECTIVE: To determine quality of communication in routine oncology consultations from patient, physician, and observer perspectives, and to determine agreement of emotional function content in consultations from these three perspectives. METHODS: In total, 69 consultations were included. Perceived quality of communication and whether or not emotional functioning had been discussed was evaluated with patient- and physician-reported questionnaires. Observer perspective was evaluated by content analysis of audio records of the consultations. Agreement between perspectives was analyzed and means compared using linear mixed models. RESULTS: The patients' ratings of communication quality differed significantly from those of both the physician and observer. Observer and physician scores did not differ significantly. Physicians rated emotional functioning as discussed more often than was reported from patient and observer perspectives. CONCLUSION: The patients' view of the quality of communication differed from that of the physician and observer. Whether emotional functioning was discussed or not was also perceived differently by patients, physicians, and observer. PRACTICE IMPLICATIONS: The underpinnings and implications of these results need to be further explored regarding how to move toward a higher degree of shared understanding, where different perspectives are more in alignment, and how to develop more valid methods for evaluating communication.

Increased levels of leukocyte-derived MMP-9 in patients with stable angina pectoris.

OBJECTIVE: There is a growing interest for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in plasma as novel biomarkers in coronary artery disease (CAD). We aimed to identify the sources of MMP-8, MMP-9, TIMP-1 and TIMP-2 among peripheral blood cells and further explore whether gene expression or protein release was altered in patients with stable angina pectoris (SA). METHODS: In total, plasma MMP-9 was measured in 44 SA patients and 47 healthy controls. From 10 patients and 10 controls, peripheral blood mononuclear cells (PBMC) and neutrophils were isolated and stimulated ex vivo. MMPs, TIMPs and myeloperoxidase were measured in plasma and supernatants by ELISA. The corresponding gene expression was measured by real-time PCR. RESULTS: Neutrophils were the dominant source of MMP-8 and MMP-9. Upon moderate stimulation with IL-8, the neutrophil release of MMP-9 was higher in the SA patients compared with controls (p<0.05). In PBMC, the TIMP-1 and MMP-9 mRNA expression was higher in SA patients compared with controls, p<0.01 and 0.05, respectively. There were no differences in plasma levels between patients and controls except for TIMP-2, which was lower in patients, p<0.01. CONCLUSION: Measurements of MMPs and TIMPs in plasma may be of limited use. Despite similar plasma levels in SA patients and controls, the leukocyte-derived MMP-9 and TIMP-1 are significantly altered in patients. The findings indicate that the leukocytes are more prone to release and produce MMP-9 in symptomatic and angiographically verified CAD-a phenomenon that may have clinical implications in the course of disease.

Lipoxin A4 inhibits porphyromonas gingivalis-induced aggregation and reactive oxygen species production by modulating neutrophil-platelet interaction and CD11b expression.

Porphyromonas gingivalis is an etiological agent that is strongly associated with periodontal disease, and it correlates with numerous inflammatory disorders, such as cardiovascular disease. Circulating bacteria may contribute to atherogenesis by promoting CD11b/CD18-mediated interactions between neutrophils and platelets, causing reactive oxygen species (ROS) production and aggregation. Lipoxin A4 (LXA4) is an endogenous anti-inflammatory and proresolving mediator that is protective of inflammatory disorders. The aim of this study was to investigate the effect of LXA4 on the P. gingivalis-induced activation of neutrophils and platelets and the possible involvement of Rho GTPases and CD11b/CD18 integrins. Platelet/leukocyte aggregation and ROS production was examined by lumiaggregometry and fluorescence microscopy. Integrin activity was studied by flow cytometry, detecting the surface expression of CD11b/CD18 as well as the exposure of the high-affinity integrin epitope, whereas the activation of Rac2/Cdc42 was examined using a glutathione S-transferase pulldown assay. The study shows that P. gingivalis activates Rac2 and Cdc42 and upregulates CD11b/CD18 and its high-affinity epitope on neutrophils, and that these effects are diminished by LXA4. Furthermore, we found that LXA4 significantly inhibits P. gingivalis-induced aggregation and ROS generation in whole blood. However, in platelet-depleted blood and in isolated neutrophils and platelets, LXA4 was unable to inhibit either aggregation or ROS production, respectively. In conclusion, this study suggests that LXA4 antagonizes P. gingivalis-induced cell activation in a manner that is dependent on leukocyte-platelet interaction, likely via the inhibition of Rho GTPase signaling and the downregulation of CD11b/CD18. These findings may contribute to new strategies in the prevention and treatment of periodontitis-induced inflammatory disorders, such as atherosclerosis.

Communication analysis in oncology care. Performance of a combination of a content analysis system and a global scale.

OBJECTIVE: The aim was to assess the feasibility and reliability of Velikova's Content Analysis System (VCAS) and the Medical Interaction Process System (MIPS) global scale for evaluation of communication in oncology care. METHODS: Seventy routine physician consultations with gastro-intestinal (GI) cancer patients were audio-recorded. Two coders applied VCAS and MIPS global scale to the consultations. VCAS captures aspects of communication like symptoms, side effects, functional issues (e.g. emotional, social, physical), health-related quality of life and medical decision making. MIPS global scale measures the total impression of the consultation, e.g. patient centredness and psychosocial focus. RESULTS: In total, 61 of 70 consultations were coded. The coding took twice the consultations' actual durations in minutes for VCAS. The time for coding MIPS global scale equalled the consultations length. However, the coder had then listened to the consultation twice before, coding for VCAS. Cohen's kappa for all aspects measured by VCAS varied between 0.20 and 1, mean 0.80. One category (Info on test) had a kappa of 0.20, the other categories were all above 0.60. Weighted Kappa for MIPS global scale varied between 0.25 and 0.73, mean 0.42. CONCLUSIONS: VCAS and MIPS global scale is a feasible combination of tools for evaluating patient-physician communication regarding content, medical decision making and global aspects of communication. VCAS showed high reliability. The MIPS global scale showed lower reliability, due to its sensitivity to the individual coders' unique values, common for all global scales. Further development of the combination of content and global instruments would be valuable.

Enhanced neutrophil expression of annexin-1 in coronary artery disease.

The systemic inflammatory activity in patients with stable coronary artery disease (CAD) is associated with a dysregulated cortisol response. Moreover, an aberrant activation status of neutrophils in CAD has been discussed; and the question of glucocorticoid resistance has been raised. The anti-inflammatory actions of glucocorticoids are mediated by annexin-1 (ANXA1). We investigated the expression of glucocorticoid receptors (GR) and ANXA1, as well as the exogenous effects of ANXA1 on neutrophils in CAD patients and related the data to diurnal salivary cortisol. Salivary cortisol levels were measured in the morning and evening during 3 consecutive days in 30 CAD patients and 30 healthy individuals. The neutrophil expression of GR and ANXA1 was determined by flow cytometry. The effect of exogenous ANXA1 was determined in a neutrophil stimulation assay. The patients showed a flattened diurnal cortisol pattern compared with healthy subjects, involving higher levels in the evening. The neutrophil expression of GR-total and GR-alpha was decreased, whereas the GR-beta expression did not differ compared with controls. The neutrophil expression of ANXA1 was significantly increased in patients. Ex vivo, ANXA1 impaired the leukotriene B(4)-induced neutrophil production of reactive oxygen species in patients but not in controls. Our findings indicate a persistent overactivation of the hypothalamic-pituitary-adrenal axis in CAD patients but do not give any evidence for glucocorticoid resistance, as assessed by the neutrophil expression of GR and ANXA1. The altered neutrophil phenotype in CAD may thus represent a long-term response to disease-related activation.

Patient-physician communication during oncology consultations.

OBJECTIVE: The aim of this study was to characterize the content of patient-physician communication in standard oncology care. METHODS: The sample consisted of 19 patients with gastrointestinal cancer. The consultations were audio-recorded, transcribed verbatim, and analyzed according to qualitative content analysis. RESULTS: The analysis resulted in seven main categories: Disease and treatment, Healthcare planning, Everyday living, Psychological well-being, Coping with disease, Expressions of concerns and feelings, and Other aspects of communication. The main focus during the consultations was on disease and treatment. Physicians tended to concentrate on response to treatment and types and severity of side effects and how to treat them. More patient-centered subjects of psychosocial character like coping and psychological well-being were discussed only briefly, if at all. CONCLUSIONS: This study adds to the information given by the existing communication analysis systems, and hence we suggest a development of the psychosocial content categories of those systems to make them more valid.

Neutrophil activation status in stable coronary artery disease.

BACKGROUND: During the last years, neutrophils have emerged as important players in atherogenesis. They are highly activated in peripheral blood of patients with unstable angina. Moreover, a primed state of circulating neutrophils has been proposed in patients with stable angina. Our aim was to investigate the neutrophil activation status in patients with stable coronary artery disease (CAD) at conventional drug treatment. METHODOLOGY AND PRINCIPAL FINDINGS: Thirty patients with stable CAD and 30 healthy controls were included using a paired design. The neutrophil expression of CD18 and high-affinity state of CD11b was analysed by flow cytometry before and after stimulation with chemoattractants. Also, the production of reactive oxygen species (ROS) was determined by chemiluminescence. During basal conditions, the neutrophil expression of CD18 or high-affinity state of CD11b did not differ between patients and controls. Chemoattractants (Interleukin-8 and Leukotriene B(4)) did not increase either the expression or the amount of high-affinity CD11b/CD18-integrins in CAD patients compared to controls, and had no effect on the production of ROS. On the other hand, the ROS production in response to C3bi-opsonised yeast particles and the neutrophils' inherent capacity to produce ROS were both significantly decreased in patients. CONCLUSION/SIGNIFICANCE: We could not find any evidence that neutrophils in patients with stable CAD were primed, i.e. more prone to activation, compared to cells from healthy controls. According to our data, the circulating neutrophils in CAD patients rather showed an impaired activation status. It remains to be elucidated whether the neutrophil dysfunction in CAD is mainly a marker of chronic disease, an atherogenic factor or a consequence of the drug treatment.