Mediterranean Diet and sub-clinical chronic inflammation: the MOLI-SANI Study

Send Orders for Reprints to reprints@benthamscience.ae Endocrine, Metabolic & Immune Disorders - Drug Targets, 2015, 15, 000-000 Mediterranean Diet The Moli-sani Study and Low-grade Subclinical 1 Inflammation: Marialaura Bonaccio*, Chiara Cerletti, Licia Iacoviello and Giovanni de Gaetano Department of Epidemiology and Prevention, IRCCS Istituto Neurologico Mediterraneo NEUROMED, Pozzilli (IS), Italy Abstract: Low-grade chronic inflammation is an underlying pathophysiological mechanism linking risk factors and/or metabolic disorders to increased risk of chronic degenerative disease. A meat-based pattern, as the Western type diet, is positively linked to higher levels of some important biomarkers of inflammation, such as C-reactive protein (CRP), interleukin-6 and fibrinogen. Conversely, a Mediterranean-like eating behavior is associated with lower degree of these biomarkers thus suggesting an anti-inflammatory action of its main food components. This chapter goes through the most important investigations addressing the relationship between dietary habits and subclinical inflammation. Attention was focussed on the findings from the Moli-sani study: this is a large prospective cohort study that recruited 24,325 men and women from the general population of the Molise Region, a Southern Italian area, with the aim of investigating genetic and environmental risk/protection factors for cardiovascular and tumor disease. For the first time, the Moli-sani study carefully investigated the Mediterranean diet as an environmental determinant of both platelet and white blood cell counts, starting from the hypothesis that a diet rich in healthy compounds could favorably influence the production and/or the clearance of these two cellular biomarkers of low-grade inflammation. Additionally, evidence from this large Italian cohort showed that a Mediterranean-like diet was closely associated with relatively lower values of glucose, lipids, CRP, blood pressure and 10-year cardiovascular risk, while the consumption of healthy foods with high rather than low content in antioxidant vitamins and phytochemicals was associated with lower blood pressure and CRP plasma levels at least in men. Keywords: Antioxidants, biomarkers of inflammation, cardiovascular disease, cancer, low-grade inflammation, mediterranean diet. BIOMARKERS OF INFLAMMATION CHRONIC LOW-GRADE Low-grade inflammation is a condition not yet uniformely defined: several biomarkers and different levels of their increase have been reported to be associated with a condition of low-grade inflammation [1]. Here a low-grade inflammation is intended as a subclinical condition (systemic or local, often chronic) characterized by increased levels of plasmatic and/or cellular biomarkers of inflammation (e.g. C-reactive protein, platelet or leukocyte counts) without any apparent clinical sign. In the absence of an acute inflammatory condition (e.g. CRP <10 mg/L or WBC <=10 x109/L), a low-grade inflammation may reflect different degrees of variance within the normal range of each specific biomarker. A low-grade inflammation has also been defined as an underlying pathophysiological mechanism linking risk factors or metabolic disorders (as oxidative stress, obesity, diabetes, dyslipidemia, etc.) to an increased risk of chronic degenerative disease [1]. C-reactive protein is the beststudied marker of subclinical inflammation [2] but several other inflammatory biomarkers have been associated with *Address correspondence to this author at the Department of Epidemiology and Prevention, IRCCS Istituto Neurologico Mediterraneo NEUROMED, Pozzilli (IS), Italy, Via dell'Elettronica, 86077 POZZILLI (IS), Italy; Tel: +39.0865.929.653; Fax: +39.0865.927.575; E-mail: marialaura.bonaccio@moli-sani.org 1871-5303/15 $58.00+.00 low-grade inflammation. Increased levels of interleukins, fibrinogen and the adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular adhesion protein-1 (VCAM-1), have been shown to be directly associated and to predict type-2 diabetes, cardiovascular disease and cancer; conversely, levels of anti-inflammatory biomarkers, such as adiponectin, have been associated with the reduction of cardiovascular disease, type-2 diabetes, and obesity-related cancer [1]. A fatty meal may represent an acute in vivo challenge that induces a low-grade inflammatory reaction. In a group of subjects at different degree of cardiovascular risk, the post-prandial condition, characterized by acutely elevated levels of triglyceride-rich lipoproteins, was accompanied by significant changes in haematological parameters: increased white blood cell counts, in particular granulocytes, increased platelet count and decreased mean platelet volume [3, 4]. The changes were statistically significant, although in a small range of differences, and were accompanied by the cell surface expression of activation markers and myeloperoxidase degranulation within granulocytes. The post-prandial state, a physiological phenomenon that may occur several times a day, could lead to a complex series of events (including endotoxemia) that may initiate and develop atherothrombosis. The occurrence of similar predictive biomarkers in conditions and diseases being apparently different for their pathogenesis, such as vascular diseases and cancer, together © 2015 Bentham Science Publishers 2 Endocrine, Metabolic & Immune Disorders - Drug Targets, 2015, Vol. 15, No. 1 Bonaccio et al. with additional pharmacological and epidemiological evidence, supports the hypothesis of a common soil underlying the pathogenetic mechanisms involved [5]. frequency questionnaire [17, 18] was administered to all subjects, allowing analyses of diet and dietary components association with different biomarkers and incidence of diseases at follow-up. MEDITERRANEAN DIET AND CHRONIC INFLAMMATION One of the very first findings from this large communitybased study was the relationship between different dietary patterns and well-known markers of inflammation or risk factors [19]. The so-called a posteriori approach was used in this type of analysis, a data-driven way which basically allows to put together a list of specific food groups in a way reflecting the actual dietary habits of the study population. This approach differs from the a priori scores which are more oriented to test whether the eating behavior of people does reflect a pre-defined ideal way of eating. LOW GRADE The relationship between low-grade inflammation and dietary habits has been investigated by either single nutrient or food approaches but recent evidence suggests that the diet as a whole is more effective in accounting for the beneficial health outcomes documented [6]. Epidemiological studies support the evidence of an association between dietary patterns and biomarkers of inflammation, as summarized in the recent systematic review by Barbaresko and coworkers [1]. A meat-based pattern, as the Western type diet, is positively linked to higher levels of some important biomarkers of inflammation, such as CRP, interleukin-6 and fibrinogen. Conversely, a Mediterranean-like eating behavior is associated with lower degree of these biomarkers thus suggesting an anti-inflammatory action of its main food components. The traditional Mediterranean diet (MD) was originally defined by the American scientist Ancel Keys who firstly described the dietary behavior of the Mediterranean people living in Southern Italy whose consumption of some particular foods was more frequent as compared to other populations [7]. Moreover, the number of cardiovascular deaths within that Mediterranean population was much lower than that recorded in the United States [8] and vital statistical data were confirmatory, e.g., Italian CHD death rates for middle-aged men in 1960 being less than half that for U.S. men [9]. These facts led Keys to reason for a possible link between dietary behavior and risk of coronary heart disease. The MD is characterized by a wide consumption of plant foods, cereals, legumes, fish, nuts, olive oil as the main fat sources and moderate wine consumption [10, 11]. Since Keys’ first investigation, a large body of evidence has linked the MD to reduced risk of major chronic diseases, such as cardiovascular and cerebrovascular disease, tumours and neurodegenerative disease [12]. The beneficial effects of MD on health have been basically ascribed to its high content of antioxidants, fibre, monounsaturated and polyunsaturated fatty acids (PUFA) [13]. In particular, antioxidants and polyphenols have been shown to exert a positive role against ischemic vascular disease mainly due to their anti-inflammatory properties [14, 15]. PEOPLE EAT FOODS NOT NUTRIENTS: MEDITERRANEAN DIET AS A WHOLE Moli-sani is a prospective cohort study which has recruited 24,325 men and women from the general population of the Molise Region, a Southern Italian area, with the aim of investigating genetic and environmental risk/protection factors for cardiovascular and tumor diseases [16]. In this study, the Italian version of the EPIC food In the study by Centritto et al. [19], the following three dietary patterns generated by principal component analysis (PCA) were identified. A “Mediterranean-type” pattern was characterized by high combined intake of olive oil, vegetables, legumes, soups, fruits and fish, and was basically comparable to what is commonly defined as a MD-style. A second pattern was the “Western-type” characterized instead by high consumption of pasta with tomato sauce, red meat, animal fats and alcohol and was somehow considered as an Italian version of the Western-type diet most popular in Western countries. An “Eggs and Sweets” pattern presented positive loadings of eggs, processed meat, margarines, butter, sugar and sweets. These three dietary models were then analysed in relation to a wide panel of well recognized markers of inflammation and to a series of risk factors for major chronic diseases. The Mediterranean-type eating behavior was closely associated with relatively lower values of glucose, lipids, Creactive protein, blood pressure and cardiovascular risk. Conversely, subjects adhering most to the Western-type pattern showed higher glucose, lipids, C-reactive protein and cardiovascular risk score, whereas the Eggs and Sweet type had significantly higher levels of C-reactive protein. Similar findings from the ATTICA study [20] reveal that levels of some crucial markers of inflammation and coagulation were significantly lower in healthy adults reporting higher adherence to the MD. The group sticking the most to this pattern reported lower levels of CRP, IL-6, homocysteine and fibrinogen and lower white blood cell count as compared with those in the lowest adherence group. Possible mechanisms underlying the beneficial effects deriving from following a Mediterranean-like diet have been ascribed to the high antioxidant content of the foods typical of this eating behavior. The protective role of antioxidant compounds, included in a healthy dietary pattern such as the Mediterranean diet, on blood pressure levels and hypertension has been established in epidemiological studies and clinical trials [21, 22]. For instance, vegetarians, whose antioxidant vitamins and phytochemicals consumption is particularly high, tend to have lower blood pressure than non- vegetarians [23]. Mediterranean Diet and Low-grade Subclinical Inflammation Endocrine, Metabolic & Immune Disorders - Drug Targets, 2015, Vol. 15, No. 1 A study considered within the Moli-sani cohort provides further support to the role played by dietary antioxidants [24]. In a total of 6,879 women and 6,892 men from the cohort, the consumption of healthy foods with high rather than low content in antioxidant vitamins and phytochemicals was associated with lower blood pressure and CRP plasma levels in men, whereas these associations were not apparent in women. In men, an increase in the food antioxidant content, which represents an increased consumption of high antioxidant content with respect to low antioxidant content foods, was associated with a decrease in systolic and diastolic blood pressure and CRP levels [24]. The possible greater protective role of healthy foods with high as compared with low antioxidant content foods on hypertension and inflammation stresses the importance of studying healthy foods according to their content in antioxidant vitamins and phytochemicals, in primary prevention of cardiovascular disease [24]. INTERVENTION STUDIES WITH MEDITERRANEAN DIET The relationship between diet and inflammatory markers has also been investigated in intervention studies. In a small trial performed on Swedish subjects, Ambring and colleagues [25] tested whether a Mediterranean-inspired diet provided to healthy subjects would affect either the inflammatory process, endothelial indexes such as vasoregulation and vascular endothelial growth factor (VEGF) or serum phospholipid fatty acid composition. A total of 22 subjects (10 women) received a Mediterraneaninspired diet or an ordinary Swedish diet for 4 weeks in a crossover way. The Mediterranean-inspired diet was able to reduce the number of leukocytes and platelets by 10% and 15%, respectively, and VEGF levels by about 15%. Neither C-reactive protein nor IL-6 concentrations were changed significantly and this has been ascribed to a relatively low degree of vascular inflammation in these healthy subjects [25]. Consistently, in a similar intervention study in patients with metabolic syndrome and higher CRP concentrations, the Mediterranean diet improved endothelial dysfunction and reduced markers of vascular inflammation [26]. The observed positive outcomes were attributed to the higher concentrations of n-3 fatty acids, which promote a favorable composition of phospholipids and are largely present in seafood and nuts. Recent studies turned attention to subclinical inflammation in adolescents, with particular regard to obese kids for whom the adipose tissue represents a favorable condition for a lowgrade inflammation to occur [27]. In 12- to 17-year old Spanish girls’ measures of adiposity (BMI and waist to hip ratio) were significant predictors of plasma levels of markers of inflammation such as leptin and hs-CRP [28] whereas concentrations of adiponectin were positively linked to a Mediterranean dietary pattern. Although, the observed relationship was mainly mediated by fat mass (BMI) and body fat distribution (WtHR), the diet may modulate the association between adiposity and subclinical inflammation. The relatively poor accounting of diet may be ascribed to the fact that the association between nutrients and inflammation has been usually investigated 3 within populations affected by disease or at high risk of certain diseases. Studies in healthy adults are scarce and just lately this research question has been addressed in largescale populations of healthy adolescents. Indeed, this study is of importance since is one of the first addressing the relationship between subclinical inflammation and diet in adolescent healthy subjects. INSIDE THE MEDITERRANEAN DIET: ANY ROLE OF SINGLE FOODS FROM THE PYRAMID? Although MD should be considered as a whole, a “single food approach” was often used to test possible associations with low-grade inflammation, in apparently healthy individuals or in subjects at different cardiovascular risk. In these studies, beside the traditional Mediterranean dietary components, other polyphenol and flavonoid-rich foods (and beverages) such as cocoa, coffee and tea have been investigated. Coffee, tea and chocolate seem to exert their optimal favorable effects on cardiovascular risk profile following a regular and moderate consumption, while healthy outcomes vanish at large consumption [29]. A study by Di Giuseppe et al. [30] has shown the beneficial properties of intake of dark chocolate against lowgrade inflammation, defined as high plasma levels of CRP. A J-shaped relationship between dark chocolate consumption and serum CRP was observed: consumers of up to 1 serving (20 g) of dark chocolate every 3 days had serum CRP concentrations significantly lower than either nonconsumers or higher consumers. Another food item that has attracted great scientific interest is red wine, a rich source of antioxidant and antiinflammatory polyphenols. In a prospective randomized crossover study by Estruch and coworkers [31], the different effects of red wine and polyphenol-free gin consumption were compared. Forty healthy men consumed 30 g of ethanol per day as either wine or gin for 28 days. Both wine and gin showed anti-inflammatory effects by reducing plasma fibrinogen and IL-1alpha levels. However, wine had the additional effect of decreasing hs-CRP (-21%), VCAM-1 (-17%) and ICAM-1 (-9%), as well as monocyte and endothelial adhesion molecules. In another study, regular wine consumption was able to prevent acute toxicity in patients undergoing radiotherapy after conservative surgery for breast carcinoma. The daily amount of alcohol intake influenced the incidence of skin toxicity, with an incidence of 38.4% in patients with no wine intake, 31.8% in patients drinking half a glass per day, 13.6% in patients drinking one glass per day, and 35.0% in patients drinking two glasses per day [32]. RECENT INSIGHTS FROM THE MOLI-SANI STUDY: MEDITERRANEAN DIET, WHITE BLOOD CELL AND PLATELET COUNTS A direct association between white blood cell count and increased morbidity and mortality for ischemic vascular disease has been observed for more than half a century [33]. From numerous epidemiological studies and meta-analyses the risk of coronary heart disease, and of different end- 4 Endocrine, Metabolic & Immune Disorders - Drug Targets, 2015, Vol. 15, No. 1 Bonaccio et al. points, was significantly greater in subjects with higher WBC count than in those with lower WBC count (all values being within the normal range) [34]. trends across the categories of coronary risk after adjustments (for age, gender, race, BMI, and various comorbidities). The Baltimore longitudinal study investigated the secular trend in WBC count and its relationship with mortality between 1958 and 2002: a downward trend, independent of age, gender, race, smoking, BMI, physical activity, was linearly associated with cardiovascular mortality and nonlinearly with all-cause mortality; participants with > 6,000 WBC/µL had higher mortality than those with 3,500-6,000 cells/uL [35]. Patients undergoing percutaneous coronary interventions in the higher tertile of platelet count measured at baseline reported a small but significant increase in the risk of 30-day mortality, in respect to patients in the lowest tertile. However, the 30-day incidence of major adverse cardiac events did not differ significantly according to tertiles of platelet count [43]. A high WBC count also predicts greater risk of reinfarction and of in-hospital death after coronary bypass graft [36]. In the Lyon Diet Heart Study, a secondary prevention trial, the number of leukocytes was shown to be linked to cardiovascular disease risk [37]. Also in conditions of leukocytosis as in patients with polycytemia vera, WBC count was directly associated with major thrombotic risk, with hazard ratios of 1.71 (1.10-2.65) and 2.84 (1.25-6.46) for risk of thrombosis and myocardial infarction, respectively, in subjects with leukocyte count >15x 109/L vs. <10x 109/L [38]. The predictive value of WBC count is plausible as these cells make a major contribution to the rheologic properties of blood, alter adhesive properties under stress, including the stress of ischemia, enhancing their rheologic importance, participate in endothelial injury, both acutely and chronically, by adhering to endothelium and damaging it with toxic oxygen compounds and proteolytic enzymes, interact with platelets, contributing to their activation and release of bioactive compounds. It will therefore be interesting to verify in future studies whether conditions that modify the leukocyte number, may also affect other markers of low grade chronic inflammation. At variance with leukocyte count, a very scarce epidemiological evidence is available on platelet count and risk of vascular disease, although inhibition of platelet function activation is effective in preventing secondary cardiovascular events [39]. Platelet count has been associated with vascular and nonvascular death, including cancer [39, 40]. More recently, precancer platelet count was found to be significantly associated with risk of symptomatic venous thromboembolism in cancer patients, but not in cancer-free subjects leading to speculate that platelet count and platelet-leukocyte interactions may have a role in the pathogenesis of cancer-related venous thromboembolism [41]. Park et al. [42] in a large representative sample of the United States population, identified a positive, independent, and dose-dependent relation between systemic inflammatory markers and the degree of estimated 10-year risk for coronary artery disease, categorized according to the Framingham risk score. Besides inflammatory markers already identified (CRP, fibrinogen, homocysteine and leukocytes), platelet levels showed significantly positive In the Moli-sani study, white blood cell count has been shown to be one of the main determinants of platelet count variability, together with CRP and D-dimer levels, independent of age and sex [44]. Starting from this original observation and from the recognized role of platelets in inflammation, researchers from the Moli-sani study carefully investigated the association of a Mediterranean diet with platelet or white blood cell counts based on the hypothesis that a diet rich in healthy compounds could favourably influence these two cellular biomarkers of low-grade inflammation in subjects without any overt chronic or hematological disease [45]. Platelet and WBC ranges were set according to normal values recently established for the Italian population [46]; this means that low or high platelet or WBC categories did not indicate any pathological value (Table 1). Results clearly showed that a better adherence to a Mediterranean dietary pattern was significantly associated with reduced WBC and platelet counts, independently of other known inflammatory markers such as CRP (Fig. 1). Moreover, subjects reporting a higher adherence to this eating pattern revealed reduced odds of being in the highest platelet count group compared to those with lower adherence. Similarly, maximum adherence to MD was also linked to greater chances of having low WBC count. The associations observed between diet and the levels of these two inflammatory cells were partially explained by the high content of both antioxidants and fiber typical of a Mediterranean-style behavior. The findings are in line with the results, already mentioned, of an intervention study showing that a MD eating pattern was directly linked to a lowering in some markers of inflammation, including platelets and WBC [23]. The favorable accounting of antioxidants is strictly related to reducing oxidative stress, an important intermediate mechanism of disease. This issue has been addressed in a well- controlled study of twins aimed at investigating the association of the Mediterranean diet and oxidative stress in which an established plasma marker of oxidative stress, namely the ratio of reduced to oxidized glutathione (GSH/GSSG), was considered. The association between the Mediterranean diet and the decreased oxidative stress was robust and not confounded by genetic or shared environmental factors and represents a plausible mechanism linking the Mediterranean diet to reduced cardiovascular disease risk [47]. The already mentioned changes in platelet and white blood cell counts induced by a standardized fatty meal [4] Mediterranean Diet and Low-grade Subclinical Inflammation Table 1. Endocrine, Metabolic & Immune Disorders - Drug Targets, 2015, Vol. 15, No. 1 5 Mean values of biomarkers of low-grade inflammation in a healthy Moli-sani population (n= 14,586) according to categories of adherence to the Mediterranean diet as measured by the Italian Mediterranean index. Adherence to the Mediterranean diet C-reactive protein (mg/L) 9 Platelets (x 10 /L) White blood cells (x 109/L) 9 Neutrophil/lymphocyte ratio (x 10 /L) Low Low-medium Medium-high High 1.41 (1.36-1.46) 1.35 (1.30-1.39) 1.36 (1.33-1.40) 1.33 (1.29-1.38) 252.0 (62.4) 252.1 (64.4) 249.0 (62.0) 246.8 (59.9) 6.33 (1.72) 6.31 (1.97) 6.18 (1.55) 6.15 (1.56) 2.11 (1.48) 2.05 (0.83) 2.00 (0.78) 1.98 (0.75) Values are reported as means± standard deviation and are controlled for age and sex. C-reactive protein is reported as geometric means with corresponding 95% confidence intervals. All p values<0.05 (Elaborated from data reported in reference 45). Fig. (1). Each line (vertical axes) indicates mean platelet or leukocyte counts, respectively. Categories of adherence to the Mediterranean diet as measured by the Mediterranean diet score are reported in the horizontal axis. There was an inverse relationship between adherence to this eating pattern and the number of both platelets and white blood cells (Elaborated from data reported in reference 45). were in the ranges of the cell count variations observed in concomitance with different degrees of adhesion to the Mediterranean diet. Whether the Mediterranean diet could modulate the consequences of the oxidative stress, due to an occasional fatty meal intake, remains to be established by appropriate studies. It was recently observed that orange juice consumption by healthy subjects during a fatty meal reduces the postprandial low-grade inflammatory response [48] by blunting the leukocyte count increase and myeloperoxidase release occurring after a fatty meal ingestion. This is suggestive and encouraging towards possible mechanism(s) explaining the beneficial effects of the Mediterranean diet and its prevention or modulation of the low-grade inflammation status, present in subjects at different degree of cardiovascular risk. CONCLUSIONS Studies conducted so far have shown that different diets are strictly linked to different degrees of low-grade inflammation. Dietary behaviors may act by modulating the levels of several inflammatory markers, from the most wellknown such as C-reactive protein, interleukin-6 or leukocytes, to the newest one as the platelet count. There is an ample consensus on the favorable effect of a Mediterranean-type eating, rich in antioxidants, fiber and healthy fats, on reducing these inflammatory biomarkers. At variance, Western-like or meat-based diets provided detrimental health-outcomes possibly due to their direct link to increased low-grade chronic inflammation. As mentioned above, future prospective studies should shed light on 6 Endocrine, Metabolic & Immune Disorders - Drug Targets, 2015, Vol. 15, No. 1 whether dietary patterns may yield positive changes in biomarkers of inflammation over time. [15] CONFLICT OF INTEREST DISCLOSURES None of the authors has a personal or financial conflict of interest. ACKNOWLEDGEMENTS The enrolment phase of the Moli-sani Project was performed at the Research Laboratories, Catholic University, Campobasso, Italy and was supported by unrestricted research grants from Pfizer Foundation (Rome, Italy) and the Italian Ministry of University and Research (MIUR, Rome, Italy)–Programma Triennale di Ricerca, Decreto no.1588. Pfizer Foundation and MIUR had no role in study design, collection, analysis, and interpretation of data and in the writing of this review. All Authors were and are independent from funders. The MOLI-SANI research group thanks IL Laboratory (Milano) for their generous supply of instruments and reagents. [16] [17] [18] [19] [20] [21] REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] Barbaresko J, Koch M, Schulze MB, Nöthlings U. (2013). Dietary pattern analysis and biomarkers of low-grade inflammation: a systematic literature review. Nutr Rev. 71, 511-527. Brooks GC, Blaha MJ, Blumenthal RS. Relation of C-Reactive Protein to Abdominal Adiposity. Am J Cardiol 2010;106:56 - 61. Ursini F, Sevanian A. (2002). Postprandial oxidative stress. Biol Chem. 383, 599-605. Tamburrelli C, Gianfagna F, D'Imperio M, De Curtis A, Rotilio D, Iacoviello L, de Gaetano G, Donati MB, Cerletti C. (2012) Postprandial cell inflammatory response to a standardised fatty meal in subjects at different degree of cardiovascular risk. Thromb Haemost. 107,530-537. Donati MB. (2010). The "common soil hypothesis": evidence from population studies? Thromb Res. 125, Suppl 2, S92-S95. Hu FB. (2002) Dietary pattern analysis: a new direction in nutritional epidemiology. Curr Opin Lipidol. 13, 3-9. Keys A. (1980) Seven countries. A multivariate analysis of death, coronary heart disease. Cambridge, MA: Harvard University Press, pp 1-381. Menotti A, Seccareccia F, Blackburn H, Keys A. Coronary mortality and its prediction in samples of US and Italian railroad employees in 25 years within the Seven Countries Study of cardiovascular diseases. Int J Epidemiol. 1995; 24: 515-21. Stamler J. Toward a modern Mediterranean diet for the 21st century. Nutr Metab Cardiovasc Dis. 2013; 23: 1159-62. Trichopoulou A, Costacou T, Bamia C, Trichopoulos D. (2003) Adherence to a Mediterranean diet and survival in a Greek population. N Engl J Med. 348, 2599-2608. Bonaccio M, Iacoviello L, de Gaetano G, on behalf of the MOLISANI Investigators. (2012) The Mediterranean diet: the reasons for a success. Thromb Res. 129, 401-404. Sofi F, Abbate R, Gensini GF, Casini A. (2010) Accruing evidence on benefits of adherence to the Mediterranean diet on health: an updated systematic review and meta-analysis. Am J Clin Nutr. 92, 1189-1196. Bonaccio M, Di Castelnuovo A, Bonanni A Costanzo S, De Lucia F, Pounis G, Zito F, Donati MB, de Gaetano G, Iacoviello L; Molisani project Investigators. (2013) Adherence to a Mediterranean diet is associated with a better health-related quality of life: a possible role of high dietary antioxidant content. BMJ Open. 3(8). pii: e003003. Giacosa A, Barale R, Bavaresco L, Gatenby P, Gerbi V, Janssens J, Johnston B, Kas K, La Vecchia C, Mainguet P, Morazzoni P, Negri E, Pelucchi C, Pezzotti M, Rondanelli M. (2013) Cancer prevention in Europe: the Mediterranean diet as a protective choice. Eur J Cancer Prev. 22, 90-95. [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] Bonaccio et al. Scoditti E, Calabriso N, Massaro M, Pellegrino M, Storelli C, Martines G, De Caterina R, Carluccio MA. (2012) Mediterranean diet polyphenols reduce inflammatory angiogenesis through MMP9 and COX-2 inhibition in human vascular endothelial cells: a potentially protective mechanism in atherosclerotic vascular disease and cancer. Arch Biochem Biophys. 527, 81-89. Iacoviello L, Bonanni A, Costanzo S, De Curtis A, Di Castelnuovo A, Olivieri M, Zito F, M B Donati, de Gaetano, on behalf of the Moli-sani project Investigators. (2007) The MOLI-SANI Project, a randomized, prospective cohort study in the Molise region in Italy; design, rationale and objectives. Italian J Public Health. 4, 110-118. Pala V, Sieri S, Palli D, Salvini S, Berrino F, Bellegotti M, Frasca G, Tumino R, Sacerdote C, Fiorini L, Celentano E, Galasso R, Krogh V. (2003) Diet in the Italian EPIC cohorts: presentation of data and methodological issues. Tumori. 89, 594-607. Pisani P, Faggiano F, Krogh V, Palli D, Vineis P, Berrino F. (1997) Relative validity and reproducibility of a food frequency dietary questionnaire for use in the Italian EPIC centres. Int J Epidemiol. 26, (Suppl 1) S152-S160. Centritto F, Iacoviello L, di Giuseppe R, et al. (2009) Dietary patterns, cardiovascular risk factors and C-reactive protein in a healthy Italian population. Nutr Metab Cardiovasc Dis. 19, 697-706. Chrysohoou C, Panagiotakos DB, Pitsavos C, Das UN, Stefanadis C. (2004) Adherence to the Mediterranean diet attenuates inflammation and coagulation process in healthy adults: The ATTICA Study. J Am Coll Cardiol. 44, 152-158. Appel LJ, Moore TJ, Obarzanek E, Vollmer WM, Svetkey LP, Sacks FM, Bray GA, Vogt TM, Cutler JA, Windhauser MM, Lin PH, Karanja N. (1997) A clinical trial of the effects of dietary patterns on blood pressure. DASH Collaborative Research Group. N Engl J Med. 336, 1117-1124. Parslow RA, Sachdev P, Salonikas C, Lux O, Jorm AF, Naidoo D. (2005) Associations between plasma antioxidants and hypertension in a community-based sample of 415 Australians aged 60-64. J Hum Hypertens. 19, 219-226. Margetts BM, Beilin LJ, Vandongen R, Armstrong BK. (1986) Vegetarian diet in mild hypertension: a randomised controlled trial. BMJ 293, 1468-1471. Pounis G, Costanzo S, di Giuseppe R, de Lucia F, Santimone I, Sciarretta A, Barisciano P, Persichillo M, De Curtis A, Zito F, Di castelnuovo A, Sieri S, Donati MB, de Gaetano G, Iocoviello L on behalf of the Moli-sani project Investigators. (2013) Consumption of healthy foods at different content of antioxidant vitamins and phytochemicals and metabolic risk factors for cardiovascular disease in men and women of the MOLI-SANI study. Eur J Clin Nutr. 67, 207-213. Ambring A, Johansson M, Axelsen M, Gan L, Strandvik B, Friberg P. (2006) Mediterranean-inspired diet lowers the ratio of serum phospholipid n-6 to n-3 fatty acids, the number of leukocytes and platelets, and vascular endothelial growth factor in healthy subjects. Am J Clin Nutr. 83, 575-581. Esposito K, Marfella R, Ciotola M, et al. (2004) Effect of a Mediterranean-style diet on endothelial dysfunction and markers of vascular inflammation in the metabolic syndrome: a randomized trial. JAMA. 292, 1440-1446. Sbarbati A, Osculati F, Silvagni D, Benati D, Galie` M, Camoglio FS et al. (2006) Obesity and inflammation: evidence for an elementary lesion. Pediatrics. 117, 220-223. Del Mar Bibiloni M1, Maffeis C, Llompart I, Pons A, Tur JA. (2013) Dietary factors associated with subclinical inflammation among girls. Eur J Clin Nutr. 67, 1264-1270. Di Castelnuovo A, di Giuseppe R, Iacoviello L, de Gaetano G. (2012) Consumption of cocoa, tea and coffee and risk of cardiovascular disease. Eur J Intern Med. 23, 15-25. di Giuseppe R, Di Castelnuovo A, Centritto F, Zito F, De Curtis A, Costanzo S, Vohnout B, Sieri S, Krogh V, Donati MB, de Gaetano G, Iacoviello L. (2008) Regular consumption of dark chocolate is associated with low serum concentrations of C-reactive protein in a healthy Italian population J Nutr. 138, 1939-1945. Estruch R, Sacanella E, Badia E, Antúnez E, Nicolás JM, FernándezSolá J, Rotilio D, de Gaetano G, Rubin E, Urbano-Márquez A. (2004) Different effects of red wine and gin consumption on inflammatory biomarkers of atherosclerosis: a prospective randomized crossover trial. Effects of wine on inflammatory markers. Atherosclerosis. 175, 117-123. Mediterranean Diet and Low-grade Subclinical Inflammation [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] Endocrine, Metabolic & Immune Disorders - Drug Targets, 2015, Vol. 15, No. 1 Morganti AG, Di Gesù C, Panunzi S, De Gaetano A, Macchia G, Deodato F, Cece MG, Cirocco M, Di Castelnuovo A, Iacoviello L, Valentini V, Cellini N, de Gaetano G. (2009) Radioprotective effect of moderate wine consumption in patients with breast carcinoma. Int J Radiat Oncol Biol Phys. 74, 1501-1505. Ernst E, Hammerschmidt DE, Bagge U, Matrai A, Dormandy JA. (1987) Leukocytes and the risk of ischemic diseases. JAMA. 257, 2318-2324. Danesh J, Collins R, Appleby P, Peto R. (1998) Association of fibrinogen, C-reactive protein, albumin, or leukocyte count with coronary heart disease: meta-analyses of prospective studies. JAMA. 279, 1477-1482. Ruggiero C, Metter EJ, Cherubini A, Maggio M, Sen R, Najjar SS, Windham GB, Ble A, Senin U, Ferrucci L. (2007) White blood cell count and mortality in the Baltimore Longitudinal Study of Aging. J Am Coll Cardiol. 49, 1841-1850. Bagger JP, Zindrou D, Taylor KM. (2003) Leukocyte count: a risk factor for coronary artery bypass graft mortality. Am J Med. 115, 660-663. de Lorgeril M, Salen P, Martin JL, Monjaud I, Delaye J, Mamelle N. (1999) Mediterranean diet, traditional risk factors, and the rate of cardiovascular complications after myocardial infarction: final report of the Lyon Diet Heart Study. Circulation. 99, 779-785. Landolfi R, Di Gennaro L, Barbui T, De Stefano V, Finazzi G, Marfisi R,Tognoni G, Marchioli R; European Collaboration on LowDose Aspirin in Polycythemia Vera (ECLAP). (2007) Leukocytosis as a major thrombotic risk factor in patients with polycythemia vera. Blood. 109, 2446-2452 Thaulow E, Erikssen J, Sandvik L, Stormorken H, Cohn PF. (1991) Blood platelet count and function are related to total and cardiovascular death in apparently healthy men. Circulation. 84, 613-617. van der Bom JG, Heckbert SR, Lumley T, Holmes CE, Cushman M, Folsom AR, Rosendaal FR, Psaty BM. (2009) Platelet count and the risk for thrombosis and death in the elderly. J Thromb Hemost. 7, 399-405. Jensvoll H, Blix K, Brækkan SK, Hansen JB. (2014) Platelet count measured prior to cancer development is a risk factor for future symptomatic venous thromboembolism: the Tromsø study. PLoS One. 9(3): e92011. Received: 21 July, 2014 Accepted: 15 October, 2014 [42] [43] [44] [45] [46] [47] [48] 7 Park CS, Ihm SH, Yoo KD, Kim DB, Lee JM, Kim HY, Chung WS, Seung KB, Kim JH. (2010) Relation between C-reactive protein, homocysteine levels, fibrinogen, and lipoprotein levels and leukocyte and platelet counts, and 10-year risk for cardiovascular disease among healthy adults in the USA. Am J Cardiol. 105, 12841288. Iijima R, Ndrepepa G, Mehilli J, Bruskina O, Schulz S, Schömig A, Kastrati A. (2007) Relationship between platelet count and 30-day clinical outcomes after percutaneous coronary interventions Pooled analysis of four ISAR trials. Thromb Haemost 98, 852-857. Santimone I, Di Castelnuovo A, De Curtis A, Spinelli M, Cugino D, Gianfagna F, Zito F, Donati MB, Cerletti C, de Gaetano G, Iacoviello L; MOLI-SANI Project Investigators. (2011) White blood cell count, sex and age are major determinants of heterogeneity of platelet indices in an adult general population: results from the MOLI-SANI project. Haematologica. 96, 11801188. Bonaccio M, Di Castelnuovo A, De Curtis A, Costanzo S, Persichillo M, Donati MB, Cerletti C, Iacoviello L, de Gaetano G. (2014) Adherence to the Mediterranean diet is associated with lower platelet and leukocyte counts: results from the MOLI-SANI study. Blood. 123: 3037-3044. Biino G, Santimone I, Minelli C, Sorice R, Frongia B, Traglia M, Ulivi S, Di Castelnuovo A, Gögele M, Nutile T, Francavilla M, Sala C, Pirastu N, Cerletti C, Iacoviello L, Gasparini P, Toniolo D, Ciullo M, Pramstaller P, Pirastu M, de Gaetano G, Balduini CL. (2013) Age- and sex-related variations in platelet count in Italy: a proposal of reference ranges based on 40987 subjects' data. PLoS One. 8(1): e54289. Dai J, Jones DP, Goldberg J, Ziegler TR, Bostick RM, Wilson PW, Manatunga AK,Shallenberger L, Jones L, Vaccarino V. (2008) Association between adherence to the Mediterranean diet and oxidative stress. Am J Clin Nutr. 88, 1364-1370. Cerletti C, Gianfagna F, Tamburrelli C, De Curtis A, D’Imperio M, Coletta W, Giordano L, Lorenzet R, Rapisarda P, Reforgiato Recupero G, Rotilio D, Iacoviello L, de Gaetano G, Donati MB (2015). Orange juice intake during a fatty meal consumption reduces the postprandial low-grade inflammatory response in healthy subjects. Thromb. Res., in press.