Tear cytokine levels are reduced in patients treated with intravitreal injections.

PURPOSE: To investigate cytokine levels in the tear fluid of patients receiving serial intravitreal injections (IVI) with anti-vascular endothelial growth factor (anti-VEGF) for neovascular age-related macular degeneration (nAMD). METHODS: Concentrations of six cytokines (IFN-γ, IL-1ß, IL-6, IL-8, TNF and VEGF) in tears of patients receiving anti-VEGF in one eye were assayed using multiplex cytometric bead array. The fellow untreated eye served as control. Tear sampling was performed on a single occasion at a minimum of four weeks after IVI. Patients underwent a pre-IVI antisepsis protocol with povidone-iodine. RESULTS: Tear fluid from thirty patients with a mean age of 78.8 years (range 58-90) was assayed. Subjects received a median of 43.5 (range 22-106) IVI in one eye. The median level of IFN-γ was 0.33 (interquartile range (IQR) 0.22-0.52) pg/mg of total protein in injected eyes versus 0.41 (IQR 0.21-1.05) pg/mg in fellow eyes (p = 0.017). For TNF, a median level of 0.12 (IQR 0.08-0.18) pg/mg of total protein was found in injected eyes versus 0.14 (IQR 0.07-0.33) pg/mg of total protein in fellow eyes (p = 0.019). There were no differences between injected and fellow eyes regarding the levels of IL-1ß, IL-6, IL-8 and VEGF. CONCLUSION: Tear fluid in eyes receiving serial IVI with anti-VEGF and preoperative povidone-iodine antisepsis constitutes lower levels of the pro-inflammatory cytokines IFN-γ and TNF compared to fellow eyes. This provides biochemical support of previous findings of reduced signs of inflammation and healthier tear film parameters in patients treated with serial IVI.

Isolation, characterization, and fibroblast uptake of bacterial extracellular vesicles from Porphyromonas gingivalis strains.

Periodontitis is an inflammatory condition caused by bacteria and represents a serious health problem worldwide as the inflammation damages the supporting tissues of the teeth and may predispose to systemic diseases. Porphyromonas gingivalis is considered a keystone periodontal pathogen that releases bacterial extracellular vesicles (bEVs) containing virulence factors, such as gingipains, that may contribute to the pathogenesis of periodontitis. This study aimed to isolate and characterize bEVs from three strains of P. gingivalis, investigate putative bEV uptake into human oral fibroblasts, and determine the gingipain activity of the bEVs. bEVs from three bacterial strains, ATCC 33277, A7A1-28, and W83, were isolated through ultrafiltration and size-exclusion chromatography. Vesicle size distribution was measured by nano-tracking analysis (NTA). Transmission electron microscopy was used for bEV visualization. Flow cytometry was used to detect bEVs and gingipain activity was measured with an enzyme assay using a substrate specific for arg-gingipain. The uptake of bEVs into oral fibroblasts was visualized using confocal microscopy. NTA showed bEV concentrations from 108 to 1011 particles/mL and bEV diameters from 42 to 356 nm. TEM pictures demonstrated vesicle-like structures. bEV-gingipains were detected both by flow cytometry and enzyme assay. Fibroblasts incubated with bEVs labeled with fluorescent dye displayed intracellular localization consistent with bEV internalization. In conclusion, bEVs from P. gingivalis were successfully isolated and characterized, and their uptake into human oral fibroblasts was documented. The bEVs displayed active gingipains demonstrating their origin from P. gingivalis and the potential role of bEVs in periodontitis.

Change in Ocular Surface Staining during Eyelid Warming Is Related to Tear Cytokine Levels.

Purpose: To investigate the changes in the tear cytokine profile of patients with meibomian gland dysfunction (MGD) treated with eyelid warming and to correlate these changes with clinical parameters for dry eye disease (DED). Methods: Seventy patients with MGD were included and treated with the warming of eyelids. Of these, 61 still used the treatment three months after baseline, while 48 completed the whole treatment period of six months. The concentrations of 39 cytokines in the tear fluid were measured at baseline and after three and six months of treatment. All participants were examined with tests for DED, including tear film break-up time (TBUT), ocular surface staining (OSS), and the self-reporting Ocular Surface Disease Index (OSDI). Changes in cytokine concentrations were assessed from baseline to three months, from three to six months, and from baseline to six months. Correlation analyses were performed between changes in the cytokine concentrations and changes in TBUT, OSS, and OSDI during the same time intervals. Results: No significant changes were found in the concentrations of the 39 cytokines during any of the three treatment intervals. However, several correlations were detected between changes in the level of cytokines and OSS from baseline to three months of treatment. Decreasing concentrations of granulocyte chemotactic protein 2 (GCP-2/CXCL6, mean effect 2.36, p=0.042), interleukin 10 (IL-10, mean effect 1.04, p=0.045), and IL-16 (mean effect 1.36, p=0.035) were associated with decreasing OSS. Decreasing concentrations of granulocyte macrophage colony-stimulating factor (GM-CSF, mean effect -2.98, p=0.024), IL-8 (IL-8/CXCL8, mean effect -1.35, p=0.026), and macrophage migration inhibitory factor (MIF, mean effect -2.44, p=0.033) were related to increasing OSS. Conclusions: Warming of eyelids did not change the concentration of cytokines in the tear fluid of patients with MGD significantly. However, alterations in the level of several cytokines were associated with changes in the OSS. This finding indicates a close connection between tear cytokines and OSS in MGD patients treated with eyelid warming.

Cardiovascular Health Effects of Shift Work with Long Working Hours and Night Shifts: Study Protocol for a Three-Year Prospective Follow-Up Study on Industrial Workers.

There is a plausible association between shift work and cardiovascular disease (CVD), which may be due to disruption of the circadian rhythm causing hormonal changes and metabolic disturbances, resulting in high blood pressure, atherosclerosis, diabetes, and being overweight. However, few studies have investigated the association between several consecutive long work shifts, including night shifts, and risk factors for developing CVD. Moreover, knowledge is lacking on factors that may modify or enhance this suggested relationship. The study period is planned from the third quarter of 2018 to the fourth quarter of 2021, and will involve 125 industrial employees at two Norwegian enterprises producing insulation. The work schedule is either rotating shiftwork (morning, evening, night) or regular day work. At baseline, we will measure blood parameters, including markers of inflammation, lipids, and glycosylated hemoglobin. We will also collect measures of blood pressure, resting heart rate, arterial stiffness, carotid intima-media thickness, and aerobic fitness. At the end of baseline data collection, a subgroup will undergo a supervised high-intensity interval training intervention for eight weeks, initiated by the Occupational Health Service. At one-year follow-up, we repeat baseline measures with added measures of heart rate variability and additional five weeks monitoring of sleep and physical activity, and assessment of respirable dust. At the two year follow-up, we will measure CVD risk factors before and after a planned three-month shutdown in one of the studied plants. We will also assess respirable dust, monitor sleep, and compile a one-year retrospective detailed overview of working hours. A final data collection, similar to the one at baseline, will be carried out after three years. We will use a comprehensive set of methods to identify the effects of shift work with long working hours and night shifts on cardiovascular health. This will provide new knowledge on the association between early manifestations of CVD and occupational exposure to shift work. Further, we can study whether work organization such as extensive overtime, sleep loss, and dust exposure have detrimental effects, and if a three-month cease in shift work or increased physical activity will modify early manifestations of CVD.

Activated dendritic cells and monocytes in HIV immunological nonresponders: HIV-induced interferon-inducible protein-10 correlates with low future CD4+ recovery.

OBJECTIVE: To explore monocyte and dendritic cell immune responses, and their association with future CD4 gain in treated HIV patients with suboptimal CD4 recovery. DESIGN: A cross-sectional study of HIV-infected, virally suppressed individuals on antiretroviral therapy for at least 24 months; 41 immunological nonresponders (INRs) (CD4 cell count <400 cells/µl) and 26 immunological responders (CD4 cell count >600 cells/µl). Ten HIV-infected antiretroviral therapy-naive and 10 HIV-negative healthy persons served as controls. CD4 cell counts were registered after median 2.4 and 4.7 years. METHODS: Monocyte, dendritic-cell and T-cell activation and regulatory T cells (Tregs) were analyzed by flow cytometry. In INR and immunological responder subgroups matched on age and nadir CD4 cell count, upregulation of interferon-inducible protein-10 (IP-10) and indoleamine 2,3-dioxygenase in monocytes and dendritic cells and cytokines in cell supernatants were measured in vitro in peripheral blood mononuclear cells stimulated with aldrithiol-2-inactivated HIV-1. RESULTS: The INR group displayed higher spontaneous activation of both monocytes (HLA-DR) and myeloid and plasmacytoid dendritic cells (HLA-DR, CD83 and CD86) compared with immunological responders, and this was associated with increased T-cell activation (CD38HLA-DR), an effector memory T-cell phenotype and activated Tregs. The IP-10 response in monocytes after in-vitro HIV stimulation was negatively associated with prospective CD4 gain. IP-10, indoleamine 2,3-dioxygenase and cytokines levels were comparable between the groups, but inversely correlated with activated Tregs in INRs. CONCLUSION: HIV-infected individuals with suboptimal immune recovery demonstrated more activated monocytes and in particular dendritic cells, compared with patients with acceptable CD4 gain. A high level of HIV-specific IP-10 expression in monocytes may be predictive of future CD4 recovery.

Physical activity initiated by employer induces improvements in a novel set of biomarkers of inflammation: an 8-week follow-up study.

PURPOSE: We investigated the level of pro- and anti-inflammatory biomarkers before and after 8 weeks of unsupervised physical activity (PA) initiated by employer. METHODS: During autumn 2014, background data, blood samples and self-reported exercise level were collected from 76 men and 41 women in a Norwegian road maintenance company. Monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), leptin, adiponectin, p-selectin and CD40 ligand (CD40L) were analyzed. [Formula: see text] was measured in a subgroup of 50 subjects. RESULTS: With reference point of exercise ≤1 time/week, we found that participants who exercised 2-3 times/week had higher [Formula: see text] values (5.6 mL kg-1 min-1; 95% CI [1.3, 9.9]). MCP-1 was lower in those who exercised ≥ 4 times/week (-81.98 pg/ml [-142.9, -21.0]). IL-6 and p-selectin levels were lower in females who exercised ≥4 times/week (-1.04 pg/ml [-2.04, -0.03] and -13.75 ng/ml [-24.03, -3.48]). Leptin was lower in participants who exercised 2-3 times/week (-0.39 µg/ml ln [-0.68, -0.09]) and ≥4 times/week (-0.69 µg/ml ln [-1.10, -0.28]). During follow-up, [Formula: see text] increased (2.9 mL kg-1 min-1 [1.5, 4.3]), while p-selectin and CD40L decreased (-2.33 ng/ml [-3.78, -0.87] and 718.14 ng/ml [-1368, -68]). MCP-1 levels decreased among men (-32.70 pg/ml [-51.21, -14.19]). A joint analysis of all biomarkers (inversed adiponectin) showed that those who exercised ≥4 times/week at baseline had lower total levels of biomarkers and that total biomarker levels decreased during follow-up. CONCLUSIONS: Exercising several times a week was associated with less inflammation compared to exercising once a week or less. During the 8-week follow-up, total levels of biomarkers of inflammation improved.

Recovery of plasma vascular endothelial growth factor concentrations during aflibercept loading phase and after the transition to bimonthly treatment for neovascular age-related macular degeneration.

AIMS: To provide data on plasma vascular endothelial growth factor (VEGF) concentration during three consecutive monthly intravitreal aflibercept injections and after transition to bimonthly treatment in patients with neovascular age-related macular degeneration (nAMD). METHODS: Sixteen consecutive treatment-naïve Caucasian patients with nAMD were included in the study. The treatment consisted of one intravitreal aflibercept (2 mg) injection every 28 days for three consecutive months followed by a fourth injection 8 weeks later. VEGF plasma concentrations were measured with Luminex on day 0 (baseline, prior to first injection); days 1, 6 and 27 (prior to second injection); day 55 (prior to third injection) and days 97 and 111 (after third injection). RESULTS: Baseline plasma VEGF concentration was 59.6±13.3 pg/mL. Aflibercept decreased plasma VEGF concentration to 32.5±3 pg/mL on day 1 (p<0.0001) and 34.7±6.3 pg/mL on day 6 (p<0.0001). On day 27, the VEGF plasma level increased to 50.6±6.5 pg/mL (p=0.009) and on day 55 to 52.8±8.8 pg/mL (p=0.027). There was no statistically significant difference between mean plasma VEGF concentrations on days 27 and 55 (p=0.139). Plasma VEGF concentration recovered completely 6 weeks after the third injection, reaching 57.9±9.6 pg/mL on day 97 (p=0.600) and 59.5±11.6 pg/mL on day 111 (p=0.987). CONCLUSIONS: Intravitreal aflibercept decreases plasma VEGF concentration mostly in the first week after treatment. Despite repeated monthly intravitreal injections, there was a monthly increase in plasma VEGF values to near baseline levels, with complete recovery 6 weeks after the third injection. TRIAL REGISTRATION NUMBER: Identifier no. NCT02125864.

Cardiomyocytes from postinfarction failing rat hearts have improved ischemia tolerance.

Altered myocardial Ca(2+) and Na(+) handling in congestive heart failure (CHF) may be expected to decrease the tolerance to ischemia by augmenting reperfusion Ca(2+) overload. The aim of the present study was to investigate tolerance to hypoxia-reoxygenation by measuring enzyme release, cell death, ATP level, and cell Ca(2+) and Na(+) in cardiomyocytes from failing rat hearts. CHF was induced in Wistar rats by ligation of the left coronary artery during isoflurane anesthesia, after which cardiac failure developed within 6 wk. Isolated cardiomyocytes were cultured for 24 h and subsequently exposed to 4 h of hypoxia and 2 h of reoxygenation. Cell damage was measured as lactate dehydrogenase (LD) release, cell death as propidium iodide uptake, and ATP by firefly luciferase assay. Cell Ca(2+) and Na(+) were determined with radioactive isotopes, and free intracellular Ca(2+) concentration ([Ca(2+)](i)) with fluo-3 AM. CHF cells showed less increase in LD release and cell death after hypoxia-reoxygenation and had less relative reduction in ATP level after hypoxia than sham cells. CHF cells accumulated less Na(+) than sham cells during hypoxia (117 vs. 267 nmol/mg protein). CHF cells maintained much lower [Ca(2+)](i) than sham cells during hypoxia (423 vs. 1,766 arbitrary units at 4 h of hypoxia), and exchangeable Ca(2+) increased much less in CHF than in sham cells (1.4 vs. 6.7 nmol/mg protein) after 120 min of reoxygenation. Ranolazine, an inhibitor of late Na(+) current, significantly attenuated both the increase in exchangeable Ca(2+) and the increase in LD release in sham cells after reoxygenation. This supports the suggestion that differences in Na(+) accumulation during hypoxia cause the observed differences in Ca(2+) accumulation during reoxygenation. Tolerance to hypoxia and reoxygenation was surprisingly higher in CHF than in sham cardiomyocytes, probably explained by lower hypoxia-mediated Na(+) accumulation and subsequent lower Ca(2+) accumulation in CHF after reoxygenation.