Genistein effects on haematoimmune cells in a newly developed alternative toxicological model

Unexpected, sometimes opposite effects of dietary isoflavonic phytoestrogens on immunity may suggest that classical mammalian toxicological assays are not entirely suitable for preclinical safety tests of these compounds. We evaluated a new alternative model of haemocytes of Egyptian cotton worm in vivo following genistein administration. Genistein induced significant changes in nucleolar morphology of haemocytes but did not influence their counts and nucleolar indices. The results indicate that genistein does not affect proliferation and differentiation of normal cells but potentiates their immuno-competence. Egyptian cotton worm larvae seem to be the new alternative biomodel for immunological screening.     

Urocortin-induced decrease in Ca2+ sensitivity of contraction in mouse tail arteries is attributable to cAMP-dependent dephosphorylation of MYPT1 and activation of myosin light chain phosphatase

Urocortin, a vasodilatory peptide related to corticotropin-releasing factor, may be an endogenous regulator of blood pressure. In vitro, rat tail arteries are relaxed by urocortin by a cAMP-mediated decrease in myofilament Ca2+ sensitivity through a still unclear mechanism. Here we show that contraction of intact mouse tail arteries induced with 42 mmol/L KCl or 0.5 micromol/L noradrenaline was associated with a approximately 2-fold increase in the phosphorylation of the regulatory subunit of myosin phosphatase (SMPP-1M), MYPT1, at Thr696, which was reversed in arteries relaxed with urocortin. Submaximally (pCa 6.1) contracted mouse tail arteries permeabilized with alpha-toxin were relaxed with urocortin by 39+/-3% at constant [Ca2+], which was associated with a decrease in myosin light chain (MLC20Ser19), MYPT1Thr696, and MYPT1Thr850 phosphorylation by 60%, 28%, and 52%, respectively. The Rho-associated kinase (ROK) inhibitor Y-27632 decreased MYPT1 phosphorylation by a similar extent. Inhibition of PP-2A with 3 nmol/L okadaic acid had no effect on MYPT1 phosphorylation, whereas inhibition of PP-1 with 3 micromol/L okadaic acid prevented dephosphorylation. Urocortin increased the rate of dephosphorylation of MLC20Ser19 approximately 2.2-fold but had no effect on the rate of contraction under conditions of, respectively, inhibited kinase and phosphatase activities. The effect of urocortin on MLC20Ser19 and MYPT1 phosphorylation was blocked by Rp-8-CPT-cAMPS and mimicked by Sp-5,6-DCl-cBIMPS. In summary, these results provide evidence that Ca(2+)-independent relaxation by urocortin can be attributed to a cAMP-mediated increased activity of SMPP-1M which at least in part is attributable to a decrease in the inhibitory phosphorylation of MYPT1.     

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.Periodontitis is one of the most prevalent inflammatory diseases in humans, the key etiologic agent being the Gram-negative anaerobic rod Porphyromonas gingivalis (54). This bacterium not only is involved in tooth loss but also may cause recurrent bacteremias and contribute to systemic disorders, such as cardiovascular disease (10, 22, 23, 39, 46, 65). P. gingivalis expresses a broad range of virulence factors, such as cysteine proteinases (gingipains), fimbriae, lipopolysaccharide (LPS), and capsular polysaccharide. Infection with the bacterium may lead to chronic inflammation in which hyperresponsive neutrophils contribute to host-mediated tissue destruction. P. gingivalis has been found in human atherosclerotic plaques (15, 27) and has been shown to promote the phenotypic switch of murine monocytes into foam cells, e.g., by inducing reactive oxygen species (ROS) generation and the oxidation of low-density lipoprotein (LDL) (31, 38, 57).We have recently reported that the exposure of human blood to P. gingivalis causes the formation of atherogenic LDL through a gingipain-mediated cleavage of apoB-100 (5). Furthermore, P. gingivalis, unlike other periodontopathic bacteria, has been shown to trigger platelet aggregation in vitro (55, 66), mainly through the interaction between bacterial gingipains and protease-activating receptors (PARs) on the platelets (49). Since platelet aggregation precedes thromboembolic events, this is an important pathogenic feature of the bacterium (1, 32).CD11b/CD18 (complement receptor 3 or Mac-1), the main β₂ integrin expressed on leukocytes, plays an important role in inflammation by promoting leukocyte adhesion and transmigration to sites of infection and by stimulating iC3b-mediated phagocytosis and cytokine production (21). In neutrophils, CD11b/CD18 binds to the platelet GPIIb/IIIa receptor via fibrinogen, thereby mediating neutrophil-platelet interaction and ROS production (11). In accordance with this, we have shown previously that platelet-leukocyte aggregation and ROS production in whole blood are mediated through selectin- and integrin-dependent interactions involving P-selectin and CD11b/CD18 (4).CD11b/CD18 requires inside-out signaling to expose and activate its high-affinity epitope and to enable ligand binding (6). P. gingivalis has been shown to induce inside-out activation of CD11b/CD18 in monocytes/macrophages (25, 29) and to upregulate the CD11b/CD18 receptors on human neutrophils via LPS (68). The P. gingivalis-induced activation of CD11b/CD18 has been most extensively studied in monocytes/macrophages, where two main signaling pathways have been implicated. First, the CD14-mediated binding of fimbriae and LPS to toll-like receptor 2 (TLR-2) stimulates CD11b/CD18 activation through a Rac1- and phosphatidylinositol 3-kinase (PI3K)-mediated pathway (26, 28, 29). Second, the bacteria can bind and activate PAR2 via gingipains, which induces CD11b/CD18 activation (34), possibly via a Rho-dependent pathway (69). Interestingly, these two pathways have been suggested to work synergistically (67). P. gingivalis also mediates platelet and neutrophil activation by acting on platelet TLR-2 and the P13K/Akt pathway (7, 35). Harokopakis and Hajishengallis (29) have shown previously that fimbriae of P. gingivalis induce CD11b/CD18 activation in human neutrophils; however, the mechanisms by which the whole bacterium interacts with CD11b/CD18 and the associated intracellular signaling in neutrophils need to be clarified.In neutrophils, Rac2 accounts for >96% of the Rac protein expressed (33, 58) and is involved in oxidase activity (13). Upon the binding of GTP, Rac and the closely related Rho GTPase Cdc42 interact with the downstream effector p21-activated kinase (PAK) (43). In human neutrophils, CD11b/CD18-mediated adhesion and phagocytosis activates Rac2 as well as Cdc42, which correlates with ROS production (9). The involvement of Rac2 in ROS generation has been demonstrated repeatedly (13), whereas Cdc42 is suggested to have an antagonistic role in oxidative activation (14).Lipoxins (LXs) are endogenously produced eicosanoids with potent anti-inflammatory and proresolving effects (41, 63). Merched et al. (44) proposed that a failure in the endogenous synthesis of LXA₄ underlies the unremitting inflammation that fuels atherosclerosis. LXA₄ functions mainly through the G protein-coupled receptor ALXR (18) and has been shown repeatedly to be protective in periodontal disease (36, 37, 61). Mouse models demonstrate that the administration of stable LXA₄ analogues significantly inhibits P. gingivalis-induced neutrophil influx, cyclooxygenase-2 expression, and prostaglandin E₂ secretion, which is done without promoting any further spreading of the infection (56). Moreover, 15-lipoxygenase transgenic rabbits, overexpressing LXA₄, show significantly diminished bone loss upon infection with P. gingivalis compared to the bone loss of control animals (64). Lipoxins also have been shown to downregulate CD11b/CD18 expression on whole-blood leukocytes (17). We have shown previously that LXA₄ modulates the inside-out activation of CD11b/CD18 in neutrophils (51), and Godson and coworkers showed that LXA₄ may influence the activation of integrins in monocytes/macrophages by modulating Rho GTPases (40, 59).In this study, we demonstrate that LXA₄ inhibits P. gingivalis-induced platelet/leukocyte aggregation and ROS production in whole blood. Furthermore, LXA₄ blocks the bacterium-induced expression and function of CD11b/CD18 on neutrophils, possibly by inhibiting Rac2 and Cdc42 signaling pathways. Interestingly, the effects of LXA₄ in P. gingivalis-induced cellular responses appear to be dependent on platelet-neutrophil interactions.     

Intraven?se Koronarangiographie mit Synchrotronstrahlung

Summary Introduction: Coronary arterty disease still remains the primary cause of death in the western industrialized world. Although the clinical value of selective coronary angiography (SCA) is beyond dispute, the associated risk of an invasive approach, the inherent costs and the necessary hospitalization have lead to the development and investigation of novel non-invasive techniques for coronary imaging. Intravenous coronary angiography (ICA) has been shown to permit non-invasive imaging of the coronary arteries. Methods: In 66 pts (80% male, age 62 (- 8.5 years) after interventional therapy/CABG operation, ICA and a SCA were carried out within a time interval of < 6 weeks. After determination of the individual circulation time, contrast media (370 mg iodine/ml, 15 ml/s, 21 ml) was injected via a sheath in the cubital vein while the patient was sitting in an upright position in a specially designed scanning chair. In two differet projections 6-8 images/patient were obtained for further image processing and evaluation. 182 target vessels had to be evaluated (LAD 55, Cfx 21, RCA 54, Grafts 52). In 50 target vessels one or more stents were implanted. Results: 182 target vessels were evaluated according to the following criteria: no stenosis, < 70%, > = 70%, occlusion. Evaluation of the ICA and SCA images was performed by two independent investigators. Due to poor image quality, 17 vessels were not evaluated. The ICA findings were compared to that of SCA. For the LAD a sensitivity of 84% (specificity 93%), for the RCA a sensitivity of 85% (specificity 97%), for the Cfx a sensitivity of 67% (specificity 90%), and for grafts a sensitivity of 85% (specificity 97%) was calculated. Conclusion: ICA proved to be a feasible and safe technique for follow-up after coronary intervention/CABG operation on an outpatient basis. Evaluation of stents and severe calcification is possible. A good image quality provided, LAD RCA and grafts can be evaluated with an acceptable sensitivity and specifity. Due to superimpositioning the low sensitivity for the Cfx has to be compensated by further image processing. Zusammenfassung Einleitung: Obwohl die Bedeutung der selektiven Koronarangiographie (SCA) in der Diagnostik der koronaren Herzkrankheit unumstritten ist, ergibt sich aus der Invasivität mit den möglichen assoziierten Komplikationen, evtl. resultierenden Folgekosten und dem in den meisten Fällen notwendigen eintägigen Krankenhausaufenthalt der Bedarf für neue, nichtinvasive Techniken im Bereich der Koronardiagnostik. Mit der intravenösen Koronarangiographie mit Synchrotronstrahlung (ICA) steht ein Verfahren zur Verfügung, welches die nichtinvasive Darstellung der Koronararterien und Stents ohne Artefakte erlaubt. Methodik: 66 Patienten (80% männlich, Alter 62 - 8.5 Jahre) nach Koronarintervention bzw. CABG-Operation wurden innerhalb von maximal 6 Wochen mittels SCA und ICA untersucht. Nach Applikation eines Kontrastmittels (370 mg Jod/ml, 15 ml/s, 30 ml) durch eine 6 F (7 F)-Schleuse in der V. brachialis wurde in 2 unterschiedlichen Projektionen 6-8 Bilder/Patient akquiriert. Zielgefäße für die Evaluation waren: LAD n = 55, Cfx n = 21, RCA n = 54, CABG n = 52. In 50 Zielgefäßen waren ein oder mehr Stents implantiert. Ergebnisse: 182 Gefäße wurden nach folgenden Kriterien beurteilt: Keine Stenose, < 70%, > 70% und Okklusion. Die Beurteilung der ICA-Befunde und der SCA erfolgte durch zwei unabhängige Untersucher. Die Ergebnisse der ICA wurden mit denen der SCA verglichen. Wegen schlechter Bildqualität konnten n = 17 Gefäße nicht beurteilt werden. Folgende Sensitivität/Spezifität für die einzelnen Koronargefäße wurden errechnet: LAD 84%/93%, RCA 85%/97%, Cfx 67%/90%, Grafts 85%/97%. Schlußfolgerungen: Es konnte gezeigt werden, daß ICA eine durchführbare und sichere Technik für das ambulante Follow-up von Patienten nach PTCA/Stentimplantation und CABG-OP darstellt. Im Gegensatz zu anderen nichtinvasiven Techniken ist die Beurteilung auch bei Stents und Kalzifikationen möglich. Bei guter Bildqualität lassen sich weitgehend überlagerungsfrei projizierbaren Gefäße (LAD, RCA, Grafts) mit akzeptabler Sensitivität und Spezifität darstellen. Die niedrige Sensitivität bei der Darstellung der Cfx aufgrund von Überlagerung durch den linken Ventrikel muß durch weitere Entwicklungen im Bereich der Bildverarbeitung kompensiert werden.     

Sleep disturbances in untreated Parkinson’s disease

Sleep abnormalities are frequently found in Parkinson’s disease (PD). However, it is unclear if they are present from the initial stages of PD. We thus aimed to assess sleep disturbances in newly diagnosed PD patients. We investigated 20 untreated PD patients using the Epworth Sleepiness Scale (ESS), the Pittsburgh Sleep Quality Index (PSQI) and the PD Sleep Scale (PDSS). Video-polysomnography and multiple sleep latency test (MSLT) were performed in 15 patients and 15 healthy controls. The ESS score was abnormally high in one patient, while short MSLT times were found in three other patients. The PSQI was higher (p < 0.05) and the PDSS lower (p < 0.001) in patients compared with controls. Video-polysomnography demonstrated a higher percentage of rapid eye movement sleep without atonia (RWA) in patients compared with controls (mean 28 vs. 2.9%, p < 0.001), whereas only one patient had clinically manifested rapid eye movement sleep behavior disorder (RBD). Interestingly, the occurrence of RWA correlated with the motor score (ρ = 0.65, p < 0.05). This study demonstrates that sleep disturbances emerge, in a proportion of patients, from the early stages of PD. RWA is a common finding while RBD is rarely present in early untreated PD.     

Positron Emission Tomography–Based Assessment of Metabolic Tumor Volume Predicts Survival after Autologous Hematopoietic Cell Transplantation for Hodgkin Lymphoma

Autologous hematopoietic cell transplantation (AHCT) is curative for 60% of patients with relapsed or refractory Hodgkin lymphoma (R/R HL). A more precise assessment of the depth of remission before AHCT may help to identify patients likely to benefit from AHCT. We aimed to determine whether positron emission tomography (PET)-based quantitative parameters of total metabolic tumor volume (TMTV), total lesion glycolysis (TLG), and maximal standardized uptake volume (SUV_max) measured before AHCT predict progression-free survival (PFS) after transplant. Pretransplant PET/computed tomography images of 96 consecutive patients with R/R HL were analyzed. Median TMTV, TLG, and SUV_max were 7.97?cm³ (range, 1.3 to 102.1), 23.7 (range, 4.0 to 813.1), and 5.23 (range, 2.7 to 23.2). Two-year PFS in patients with high TMTV (TMTVhigh; more than median; n?=?17) was only 12% (95% CI, 1% to 38%) compared with 53% (95% CI, 28% to 73%; P?=?.05) in patients with TMTVlow (lower or equal to median; n?=?17) and 63% (95% CI, 50% to 74%) in 61 patients with no metabolically active tumor (TMTV0; P?>?.01). In concordance, high TLG (>19) and SUV_max (>4.9) predicted inferior 2-year PFS. In multivariate analysis patients with TMTVhigh had a 3.5-fold higher risk of treatment failure compared with TMTV0/TMTVlow (HR, 3.49; 95% CI, 1.75 to 6.93; P?<?.01). Deauville (D)-scores of 4 to 5 before AHCT predicted worse PFS compared with D-scores of 1 to 3 (HR, 3.7; 95% CI, 1.92 to 7.28; P?<?.01). Yet, TMTV and D-scores were disconcordant in 12 subjects; 9 patients in the D4 group with TMTVlow had 2-year PFS of 44% (95% CI, 14% to 72%), which was 2-fold higher than predicted by D4 score. In conclusion, in patients with R/R HL and PET-positive residual disease, TMTVhigh can identify very poor AHCT responders. Patients with TMTVlow, TLG, and SUV_max before AHCT have similar outcomes to those without metabolically active disease.     

CD8+ Tregs revisited: A heterogeneous population with different phenotypes and properties

Regulatory T cells (Tregs) play a key role in the peripheral self-tolerance and preventing autoimmunity. While classical CD4⁺ Foxp3⁺ Tregs are well established, their CD8⁺ counterparts are still controversial in many aspects including their phenotypic identity and their mechanisms of suppression. Because of these controversies and because of only a limited number of studies documenting the immunoregulatory function of CD8⁺ Tregs in vivo, the concept of CD8⁺ Tregs is still not unanimously accepted. We propose that any T-cell subset considered as true regulatory must be distinguishable from other cell types and must suppress in vivo immune responses via a known mechanism. In this article, we revisit the concept of CD8⁺ Tregs by focusing on the characterization of individual CD8⁺ T-cell subsets with proposed regulatory capacity separately. Therefore, we review the phenotype and function of CD8⁺ FOXP3⁺ T cells, CD8⁺ CD122⁺ T cells, CD8⁺ CD28^low/− T cells, CD8⁺ CD45RC^low T cells, T cells expressing CD8αα homodimer and Qa-1-restricted CD8⁺ T cells to show whether there is sufficient evidence to establish these subsets as bona fide Tregs. Based on the intrinsic ability of CD8⁺ Treg subsets to promote immune tolerance in animal models, we elaborate on their potential use in clinics.     

Alcohol drinking and esophageal squamous cell carcinoma with focus on light-drinkers and never-smokers: a systematic review and meta-analysis

Quantification of the association between alcohol drinking and risk of esophageal squamous cell carcinoma (ESCC) is an open issue, particularly among light alcohol drinkers, never-smokers, and Asian populations, in which some high-risk polymorphisms in alcohol metabolizing genes are more prevalent. To address these issues, we conducted a systematic review and meta-analysis using 40 case-control and 13 cohort studies that reported on the risk associated with alcohol drinking for at least three levels of consumption. In studies adjusted for age, sex, and tobacco smoking, the relative risk (RR) and 95% confidence interval (CI) for the association between light alcohol drinking (≤ 12.5 g/d) and risk of ESCC was 1.38 (1.14-1.67). The association was slightly stronger in Asian countries than in other populations. The adjusted RRs (95% CIs) were 2.62 (2.07-3.31) for moderate drinking (>12.5-<50 g/d) and 5.54 (3.92-7.28) for high alcohol intake (≥50 g/d); the RRs were slightly higher in non-Asian populations. In prospective studies, the RR (95% CI) was 1.35 (0.92-1.98) for light, 2.15 (1.55-2.98) for moderate, and 3.35 (2.06-5.46) for high alcohol intakes; light drinking showed an association with ESCC in Asia (five studies) but not in other regions (three studies). Among never-smokers (nine studies), the RR (95% CI) was 0.74 (0.47-1.16) for light, 1.54 (1.09-2.17) for moderate, and 3.09 (1.75-5.46) for high intakes. This meta-analysis further corroborates the association of moderate and high alcohol intake with risk of ESCC and provides risk estimates based on multiple prospective studies. Light alcohol intake appears to be associated to ESCC mainly in studies in Asia, which suggests a possible role of genetic susceptibility factors.