Impact of diet and nutraceutical supplementation on inflammation in elderly people. Results from the RISTOMED study, an open-label randomized control trial.

BACKGROUND & AIMS: Eating habits may influence the life span and the quality of ageing process by modulating inflammation. The RISTOMED project was developed to provide a personalized and balanced diet, enriched with or without nutraceutical compounds, to decrease and prevent inflammageing, oxidative stress and gut microbiota alteration in healthy elderly people. This paper focused on the effect on inflammation and metabolism markers after 56 days of RISTOMED diet alone or supplementation with three nutraceutical compounds. METHODS: A cohort of 125 healthy elderly subjects was recruited and randomized into 4 arms (Arm A, RISTOMED diet; Arm B, RISTOMED diet plus VSL#3 probiotic blend; Arm C, RISTOMED diet plus AISA d-Limonene; Arm D, RISTOMED diet plus Argan oil). Inflammatory and metabolism parameters as well as the ratio between Clostridium cluster IV and Bifidobacteria (CL/B) were collected before and after 56 days of dietary intervention, and their evolution compared among the arms. Moreover, participants were subdivided according to their baseline inflammatory parameters (erythrocytes sedimentation rate (ESR), C-Reactive Protein, fibrinogen, Tumor Necrosis Factor-alfa (TNF-α), and Interleukin 6) in two clusters with low or medium-high level of inflammation. The evolution of the measured parameters was then examined separately in each cluster. RESULTS: Overall, RISTOMED diet alone or with each nutraceutical supplementation significantly decreased ESR. RISTOMED diet supplemented with d-Limonene resulted in a decrease in fibrinogen, glucose, insulin levels and HOMA-IR. The most beneficial effects were observed in subjects with a medium-high inflammatory status who received RISTOMED diet with AISA d-Limonene supplementation. Moreover, RISTOMED diet associated with VSL#3 probiotic blend induced a decrease in the CL/B ratio. CONCLUSIONS: Overall, this study emphasizes the beneficial anti-inflammageing effect of RISTOMED diet supplemented with nutraceuticals to control the inflammatory status of elderly individuals.

[Cellularity and extracellularity: the multi-fibrillar system: from cytoskeleton to connective tissue]. / Cellularité et extracellularité: le système multi-fibrillaire: du cytosquelette au tissu conjonctif.

This brief text aims at illustrating the interactions between connective tissue fibers and cell cytoskeleton fibers. These two networks are connected by molecular bridges at the level of the cell membrane of the cells of the connective and vascular tissues, allowing functional adjustments across the two domains, but also the transduction of forces and tensions into a biochemical alphabet. The signaling between the cell kern and its environment, but equally the other way round, from the environment to the core of the cell, depends on it.

[Suitability to the specific task of drivers suffering from hypertension]. / Idoneità alla mansione specifica degli autisti affetti da ipertensione arteriosa.

The italian legislation defines the minimal psychophysical requirements for driving licence, that is indispensabile to be employed, but leaves extensive discretion in the evaluation of the cardiovascular diseases. Hypertension, the most important risk factor for heart disease, is not considered a contraindication to drive. Uncontrolled blood pressure or target organ damage expose to acute complications that may well preclude safe driving and be the cause of crashs; therefore the role of the occupational physician is so important in the sanitary surveillance of the drivers of vehicles for transportation of people and/or goods.

[Cardiorespiratory fitness and arterial stiffness in sedentary and not sedentary hypertensive workers]. / Relazione tra VO2 e Augmentation Index in lavoratori sedentari e non, affetti da ipertensione arteriosa.

In hypertensive subjects, arterial stiffness and arterial wave early reflection are thought to be the major factor limiting cardiac reserve and exercise capacity. Aortic augmentation index (AIx) is a measure of arterial wave reflection and stiffness, and has been associated with decreased cardiorespiratory fitness. We investigated the role of physical activity at work and its effect on such association. 25 hypertensive middle-aged workers, without history of diabetes, cardiovascular disease, renal failure and inflammatory diseases, were studied. Our study confirms that AIx provides information for the prediction of VO2 peak, being also gained with a non-invasive and practical test. Inside the two groups of sedentary and non sedentary workers, the relation between AIx and VO2 peak is still significant; whereas the non sedentary group showed a worse cardiorespiratory fitness without significantly differences in arterial stiffness.

Aging and the endothelium.

One link between aging and endothelial function is the inflammatory response. On one hand, the latter shortens the biological engaged by activated leukocytes against invaders or stressing agents. On the other hand, the surveyed tissues become targets of the toxicity of reactive oxygen species, ROS. The ensuing regeneration is source of transcriptional infidelity, leading to the alteration of the repaired tissue. Hence, the toll of inflammatory stress consists in premature senescence of cell and tissues. This hypothesis is discussed in the present review, which focuses on the molecular targets relevant for cancer and degenerative diseases, both tributary to an inflammatory environment and taking advantage from the consequences of cell and tissue dysfunctions characteristic of aging. Eventually, adaptation to stress, whatever its origin -inflammatory and/or psychosocial-is discussed. Basal nitric oxide (NO) release, such as provided through moderate exercise, seems to be the most potent guardian against immune, nervous and cardiovascular over-stimulation. Tissue regeneration is also obtained by circulating endothelial progenitors able to recognize the damaged tissue. To avoid post-inflammatory alterations resulting in detrimental changes of tissues and organs, the pharmacological protection of endothelium by agents able to modulate its activation seems crucial to us.

Antioxidant betalains from cactus pear (Opuntia ficus-indica) inhibit endothelial ICAM-1 expression.

It has been suggested that some pigments would have antioxidant properties and that their presence in dietary constituents would contribute to reduce the risk of oxidative stress-correlated diseases. Among others, inflammatory response depends on redox status and may implicate oxidative stress. Vascular endothelial cells are a direct target of oxidative stress in inflammation. We have tested the impact of the free radical scavenger and antioxidant properties of betalains from the prickle pear in an in vitro model of endothelial cells. Here we show the capacity of betalains to protect endothelium from cytokine-induced redox state alteration, through ICAM-1 inhibition.

Naftidrofuryl-driven regulation of endothelial ICAM-1 involves nitric oxide.

Naftidrofuryl is a selective inhibitor of the 5-HT2 receptor expressed on human endothelial cells. This drug has been used over the years to cope with cerebral or peripheral ischemic accidents; however, no clear mechanism of action of this molecule has been highlighted to explain its vascular effects. In the present work, we demonstrate that the involvement of nitric oxide can account for the effects of naftidrofuryl. Indeed, naftidrofuryl potently inhibited the TNF-alpha-triggered increase of intercellular adhesion molecule-1 (ICAM-1) expression as well as stress fiber formation in human umbilical vein endothelial cells (HUVEC). Moreover, naftidrofuryl induced the expression of type II nitric oxide synthase (NOS II) messenger and protein, leading to a noticeable increase in nitric oxide synthesis. Furthermore, using the specific NOS II inhibitor 1400W, we verified that the observed effects of naftidrofuryl were NOS II-dependent. The biology of nitric oxide accounts for the reduction of the vasospasm associated with stroke and the strong inhibition of platelet aggregation. In conclusion, our work provides evidence for the inhibition of leukocyte recruitment by downregulation of CD54/ICAM-1, an additional key factor to be dealt with during thrombotic accidents. Importantly, it also highlights a novel NOS II-dependent mechanism of action for naftidrofuryl.

Selective activation of cervical microvascular endothelial cells by human papillomavirus 16-e7 oncoprotein.

BACKGROUND: Human papillomavirus type 16 (HPV16) is strongly implicated in the etiology of cervical cancer, with the expression of HPV16-encoded E7 oncoprotein in infected epithelial cells contributing to their malignant transformation. Although nuclear E7 interacts with several nuclear targets, we have previously shown that extracellular E7 can cause suppression of immune cell function. Moreover, cervical microvascular endothelial (CrMVEn) cells treated with E7 increase their expression of adhesion molecules. High levels of some cytokines in serum and in cervicovaginal secretions are associated with the progression of cervical cancer. In this study, we investigated the effects of extracellular E7 on cytokine production and on cytoskeleton structure of CrMVEn cells and vascular endothelial cells from different organs. METHODS: Immunocytochemical staining and flow cytometry techniques were used to detect E7 in endothelial cells incubated with purified E7 protein. Laser scanning confocal microscopy was used to study the E7-induced modification of the endothelial cytoskeleton. An enzyme-linked immunosorbent assay was performed to measure the production of two cytokines, interleukin 6 (IL-6) and interleukin 8 (IL-8), by E7-treated endothelial cells. All statistical tests were two-sided. RESULTS: Extracellular E7 was taken up by CrMVEn cells and localized to the cytoplasm. CrMVEn cells showed a statistically significant (P<.02) increase in the production of IL-6 and IL-8 after treatment with E7 compared with the controls. CrMVEn cells also produced higher levels of these cytokines than did the other endothelial cells (P<.01). E7 also induced marked alterations in the endothelial cytoskeleton of CrMVEn cells as a result of actin fiber polymerization. CONCLUSION: These findings suggest a novel mechanism by which E7, as an extracellular factor, can play a role in the progression and dissemination of cervical cancer via its selective effects on endothelial cells.

Human papillomavirus-16-E7 oncoprotein enhances the expression of adhesion molecules in cervical endothelial cells but not in human umbilical vein endothelial cells.

OBJECTIVES: E7 is one of the oncoproteins encoded by human papillomavirus-16 (HPV-16), the major etiologic factor responsible for cervical cancer. Human papillomavirus-16-E7 expressed by human uterine cervix carcinoma cells is also released in the extracellular compartment where it induces immune suppression. We investigated whether E7 was also responsible for the enhanced endothelial adhesiveness required in cancer progression. STUDY DESIGN/METHODS: We treated cervical microvascular endothelial cells (CrMVEn) and human umbilical vein endothelial cells (HUVEC) with E7, tumor necrosis factor-alpha (TNF-alpha), and hydrogen peroxide (H2O2) and measured the expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) by fluorescent-activated cell sorter analysis. RESULTS: E7 strongly induced the expression of E-selectin, ICAM-1, and VCAM-1 in CrMVEn, but not in HUVEC. Tumor necrosis factor-alpha further increased the endothelial expression of adhesion molecules in CrMVEn. Hydrogen peroxide pre-treatment resulted in an enhanced ICAM-1 and a decreased E-selectin and VCAM-1 expression. We also show indirect effects when endothelial cells were stimulated with the supernatant of E7-pretreated macrophages. CONCLUSIONS: These results show that HPV-16-E7 oncoprotein strongly induces adhesion molecules expression in organ-specific endothelial cells.

Endothelium as a pharmacological target.

Over the last few years, the increasing knowledge of the endothelium has highlighted its integral role in a number of pathologies. Endothelial cells are pivotally involved in the recruitment and adhesion of leukocytes and platelets, and they express adhesion molecules and growth factors. This review highlights the recent advances made in the understanding of the endothelium and discusses the endothelium as a potential target in a variety of diseases, including cardiovascular diseases, cancer and inflammatory diseases.

Regulation of ICAM-1/CD54 expression on human endothelial cells by hydrogen peroxide involves inducible NO synthase.

Expression of the inducible isoform of nitric oxide synthase (iNOS) is stimulated by cytokines in human epithelial cells. This work indicates that incubation of human umbilical cord endothelial cells with combinations of interleukin-1beta, tumor necrosis factor alpha, and interferon-gamma stimulated the synthesis of iNOS mRNA, as detected by reverse transcriptase-polymerase chain reaction. It is important to note that 50, 100, and 200 microM hydrogen peroxide was able to stimulate iNOS directly. Furthermore, 100 microM H2O2 enhanced synthesis of the oxidation products, nitrite (NO2-) and nitrate (NO3-) at 12 and 36 h. iNOS protein, detected by Western blot analysis, as well as L-citrulline levels, were also increased. When endothelial cell monolayers were incubated for 1 h with 100 microM H2O2 and subsequently with cytokines, iNOS mRNA was further augmented. Under the same conditions, we regularly observed an inhibition (25%) of intercellular adhesion molecule-1 (ICAM-1/CD54) expression. The latter was reversed when the NOS inhibitor N(G)-monomethyl-L-arginine was added, as shown by flow cytometry. These data suggest a specific effect of endogenous hydroperoxides on the biosynthesis and processing of the human endothelial iNOS isoform. We propose that H2O2 induces a temporary NO-dependent modulation of adhesion molecule expression to limit the tissue destruction that accompanies the vascular recruitment of leukocytes.

HPV-16 E7 but not E6 oncogenic protein triggers both cellular immunosuppression and angiogenic processes.

HPV-16 E6 and E7 oncoproteins impair the cell cycle in human uterine cervix carcinoma cells (HUCC) by acting on p53 and retinoblastoma proteins, respectively. We recently reported that E7 related into the extracellular compartment by HUCC SiHa cells could inhibit immune T-cell response to recall and alloantigens by a mechanism involving an overproduction of the immunosuppressive IFN alpha by antigen presenting cells (APCs). In this study, we found that besides E7, E6 protein and the vascular endothelium growth factor (VEGF) were released into the SiHa cell supernatants, and we further showed that extracellular E7 but not E6 oncoprotein 1) inhibits the immune cell response to recall and alloantigens, and 2) enhances the release of angiogenic cytokines, including TNF alpha, IL-1 beta and IL-6 by macrophages and/or dendritic cells. VEGF unexpectedly released by cancer cells could also contribute to angiogenesis. Thus in HUCC the same E7 oncoprotein which contributes to controlling the cancer cell cycle has the means in its extracellular configuration to contribute to microenvironmental immunosuppressive and angiogenic processes. Neutralizing anti-E7 antibodies either passively administered or induced by active immunization could represent a new immunotherapeutic endeavour to combat the immunosuppression and/or neoangiogenesis effects of extracellular E7 protein.

Glutathione peroxidase mimics prevent TNFalpha- and neutrophil-induced endothelial alterations.

Based on the assumption that glutathione peroxidase (GPx) activity might be limiting in preventing peroxide-induced impairment of endothelial regulatory functions, we studied the effect of a series of new selenium-containing GPx mimics on endothelial cells exposed to an inflammatory stress. The two compounds that have the highest GPx activity, BXT-51072 and BXT-51077, were shown to be the most efficient inhibitors of leukocyte recruitment by human umbilical vein endothelial cells (HUVEC), upon incubation with neutrophils (10-fold excess over HUVEC) and with 1 ng/ml TNF-alpha for 1 or 3.5 h. When HUVEC were pre- and cotreated with 10 microM of either compound, neutrophil adhesion and endothelial alteration were markedly inhibited, as assessed by immunoassays of myeloperoxidase and von Willebrand factor, respectively. These two GPx mimics were also found to be the most efficient inhibitors of the TNFalpha-induced endothelial expression of P- and E-selectin and of the TNFalpha- or interleukin1-induced endothelial release of interleukin-8. Our results demonstrate that GPx mimics such as BXT-51072 behave as potent antagonists of TNF-alpha and interleukin-1 through the downregulation of endothelial proinflammatory responses.

ICAM-1 and VCAM-1 expression induced by TNF-alpha are inhibited by a glutathione peroxidase mimic.

Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are respectively involved in the endothelial recruitment of neutrophils, and in that of lymphocytes or tumor cells, in response to specific signals. We have used the glutathione peroxidase (GPx) mimic BXT-51072 to assess the possibility that endogenous hydroperoxides play a role in the tumor necrosis factor-alpha (TNFalpha)-induced expression of ICAM-1 and VCAM-1 by monolayers of human endothelial cells. The GPx mimic BXT-51072 strongly inhibits the TNFalpha-induced and cycloheximide-sensitive expression of ICAM-1 and VCAM-1. It also inhibits the TNFalpha-induced reorganization of the actin network and the associated formation of stress fibers. Actin reorganization induced by cytochalasin D treatment did not inhibit ICAM-1 expression. Our results are compatible with specific and synergistic effects of endogenous hydroperoxides on the biosynthesis and processing of cell adhesion molecules and cytoskeleton components.

Blood contamination of amniotic fluid after amniocentesis in relation to placental location.

The aim of the present study was to evaluate blood contamination of the amniotic fluid collected in 20 patients undergoing a second amniocentesis performed 2 weeks after a first procedure that had failed due to Pseudomonas aeruginosa contamination of the cell cultures. Red blood cell and haemoglobin concentrations in the amniotic fluid were significantly higher in patients who had undergone a transplacental procedure compared with patients in whom the placenta was not traversed with the needle. For both groups, blood contamination of the amniotic fluid was significantly higher compared with a control group of 20 patients undergoing amniocentesis for the first time. Significant blood contamination of the amniotic fluid after amniocentesis occurs in every instance if evaluated at a "second-look' procedure; the blood contamination is higher when an anterior placenta is traversed with the needle. The clinical significance of these findings needs to be further evaluated.

[Pharmacological modulation of alterations of endothelial cytoskeleton induced by hydrogen peroxyde and by TNF-alpha]. / Modulation pharmacologique des altérations du cytosquelette endothélial induites par le péroxyde d'hydrogène et par le TNF-alpha.

New selenium containing compounds which act as mimics of glutathione peroxidase (GPx) protect vascular endothelial cells (HUVEC) from the toxicity of 140 microM hydrogen peroxide. In the absence of GPx mimic, hydrogen peroxide destroys the tightness of the cellular monolayer and transforms the actin network into compact stress fibers. The pre-treatment of the cells by 4 microM of the lead-compound BXT-51072 for 1 hours inhibits the morphological modifications induced by hydrogen peroxide. This GPx mimic can also prevent the alterations of the endothelial cytoskeleton which are induced by Tumor Necrosis Factor-alpha (TNF-alpha) and which consist in a reorganization of actin filaments with the formation of stress fibers. Fluorescent labeling of polymerized actin has been performed by means of phalloidine coupled with rhodamine. The protective effect of this antioxidant catalyst against the toxicity of hydrogen peroxide and TNF-alpha includes the maintenance of a structural configuration of the cytoskeleton which is required for the function of endothelial barrier.

[Antioxidant and anti-inflammatory protection of vascular endothelial cells by new synthetic mimics of glutathione peroxidase]. / Protection antioxydante et anti-inflammatoire des cellules endothéliales vasculaires par de nouveaux mimes synthétiques de la glutathion peroxydase.

New selenium-containing compounds behave as GPx mimics and protect endothelial cells (HUVEC) from damage upon exposure to 55 microM linoleic acid hydroperoxide or to 200 microM hydrogen peroxide. The simultaneous presence of the GPx mimic and the hydroperoxyde is not necessary, since a pre-treatment of endothelial monolayers with 1 to 10 microM of such compounds, preserves their morphology, their cell density and their longer-term viability. The compounds which are most efficient in this model of oxidative stress also protect endothelial monolayers which have been incubated with an excess (10:1) of polymorphonuclear neutrophils (PMN) and with 1 ng/ml of TNF-alpha, if such monolayers are pre- and co-treated (10 microM). They inhibit the adhesion of activated neutrophils which show-up as polymorphous and very dense particles, in the vicinity of which endothelial alterations can be seen. The inhibition of leucocyte adhesion and that of endothelial activation/alteration have been quantified by means of immunoassays of myeloperoxidase and von Willebrand factor (vWf). The lead-compound BXT-51072 is not a direct inhibitor of the NADPH oxidase of PMN. TNF-alpha alone induces the endothelial release of Interleukin-8 (Il-8) as well as the expression of P- and E-selectin. The extent and the kinetics of inhibition of such processes by compound BXT-51072 would explain several of the effects observed in the presence of PMN. The GPx mimics also inhibit the endothelial production of Il-8 which is induced by Interleukin-1 alpha. Finally, compound BXT-51072 inhibits the endothelial expression of the adhesion factor VCAM-1 which is more slowly induced by TNF-alpha. Such antioxidant catalysts therefore protect endothelial cells from the toxic effects of TNF-alpha through mechanisms which include a down-regulation of cytokines and cell-adhesion factors.

Transplacental amniocentesis: is it really a higher-risk procedure?

The aim of this study was to compare transplacental with non-transplacental amniocentesis in terms of related complications. Between January 1991 and December 1992, 4564 genetic amniocenteses were performed in 4527 patients (4491 singleton, 35 twin, and one triplet pregnancy) at 15-16 weeks of gestation. All the procedures were ultrasound-guided and performed by the same operator. In 1487 cases, an anterior placenta was traversed with the needle, whereas in 3077 cases, the needle was inserted directly into the amniotic cavity without traversing the placenta. After the exclusion of patients in whom amniotic cell culture failed or in whom an abnormal karyotype was obtained, and of patients lost to follow-up, a total of 4454 patients (98 per cent) were followed for 30 days after amniocentesis. Two spontaneous abortions occurred after a transplacental procedure, and five after a nontransplacental procedure (P = not significant). There were no episodes of amniotic fluid leakage in the first group, whereas 16 ruptures of the membranes that resolved spontaneously occurred in the second group (P < 0.01). Our data suggest that transplacental amniocentesis carries a similar abortive risk to and a lower risk of transient rupture of the membranes than non-transplacental amniocentesis and may therefore be preferred at the gestational period examined (15-16 weeks). However, the risk of feto-maternal haemorrhage, which is reported to be higher for a transplacental procedure, must be considered in the case of an anterior placenta.