The genetic history of Italians: new-insights from uniparentally-inherited markers

Genetic differences among human groups can be ascribed both to the broad-scale extents of pre-historical and historical migrations and to the fine-scale impacts of socio-cultural and geographic heterogeneity. In this thesis, the genetic information provided by uniparental markers were exploited to address different aspects of the Italian population history, by combining macro- and micro-geographic investigations at different spatial and temporal scales. To firstly assess the overall Italian variability, Y-chromosome and mtDNA markers were deeply typed in ~900 individuals from continental Italy, Sicily and Sardinia. Sex-biased patterns and contrasting demographic histories were observed for males and females. Differential European and Mediterranean contributions were invoked to explain the paternal genetic sub-structure observed in peninsular Italy, compared to the homogeneous maternal genetic landscape. If Neolithic showed to be one principal determinant of the detected paternal structure, local insights into specific Italian regional contexts highlighted the importance of Post-Neolithic contributions. Among them, migrations from the Balkans (particularly Greece) during late Metal Ages, played a relevant role in the cultural and genetic transitions occurred in Sicily and Southern Italy. On a finer geographic and temporal perspective, the more recent layers of Italian genetic history and some aspects of the gene-culture interaction were assessed by exploring the genetic variability within two “marginal populations”: Arbereshe of Southern Italy and Partecipanza in Northern Italy. The Arbereshe are Albanian-speaking communities settled in Sicily and Calabria since the end of Middle Ages. Despite sharing common genetic and cultural backgrounds, these groups revealed diverging micro-evolutionary histories, implying different founding events and different patterns of cultural isolation and local admixture. Partecipanza is an idiosyncratic institution of Medieval origin aimed at sharing and devolving collective lands. This case-study exemplified that socio-economic stratification within the same population may induce sex-biased genetic structuring and the maintenance of otherwise hidden historical genetic traces.

Identificazione di markers morfologici e sierologici di vulnerabilità della placca carotidea

Introduzione: L’indicazione alla rivascolarizzazione carotidea è comunemente posta in base alla percentuale di stenosi, alla presenza di sintomi neurologici ed alle condizioni cliniche del paziente. Una placca ad elevato potenziale embolico viene definita “vulnerabile”; la sua caratterizzazione, tuttavia, non è universalmente accettata ai fini della rivascolarizzazione. Lo scopo dello studio è indagare il ruolo del mezzo di contrasto ecografico (CEUS) nell’identificazione della placca carotidea vulnerabile. Materiali e Metodi: I pazienti sottoposti a endoarterectomia carotidea, sono stati valutati mediante TC cerebrale preoperatoria e CEUS. Le microbolle di contrasto rilevate nella placca, indicative di neovascolarizzazione, sono state quantificate in dB-E ed istologicamente valutate per cinque caratteristiche: (densità dei microvasi, spessore del cappuccio fibroso, estensione delle calcificazioni, infiltrato infiammatorio e core lipidico) il valore da 1 a 5, ottenuto in cieco, indica in grado di vulnerabilità della placca. L'ANOVA test, il test di Fisher e t Student sono stati usati per correlare le caratteristiche dei pazienti ed istologiche col valore di dB-E. Risultati: Di 22 pazienti (range 2-7.8, media 4.85 ±1.9 SD) vi era un numero più alto di sintomatici (7.40 ± 0.5) rispetto agli asintomatici (3.5 ± 1.4) (p = 0.002). Un più alto valore di dB-E si associava con la presenza di un sottile cappuccino fibroso (<200 µm, 5.96±1.5 vs. 3 ± 1,p = 0.01) ed un maggiore infiltrato infiammatorio (3.2 ± 0.9 vs. 6.4 ± 1.2, p = 0.03). Placche con vulnerabilità 5 si associavano ad un valore più alto di dB-E rispetto alle placche con vulnerabilità 1 (7.6 ± 0.2 vs. 2.5 ± 0.6, rispettivamente, p=0.001). Preoperatoriamente, le lesioni emboliche ipsilaterali alla TC, correlavano con un più alto valore di dB-E (5.96±1.5 vs. 3.0±1.0, p=0.01). Conclusioni: Il valore di dB-E alla CEUS indica l’estensione della neovascolarizzazione della placca carotidea e può essere utilizzato come marker di vulnerabilità della placca.

Interactions between the gut microbiota, short-chain fatty acids and the immune system in pediatric patients undergoing hematopoietic stem cell transplantation

The gut microbiota (GM) is essential for human health and contributes to several diseases; indeed it can be considered an extension of the self and, together with the genetic makeup, determines the physiology of an organism. In this thesis has been studied the peripheral immune system reconstitution in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (aHSCT) in the early phase; in parallel, have been also explored the gut microbiota variations as one of the of primary factors in governing the fate of the immunological recovery, predisposing or protecting from complications such as the onset of acute graft-versus-host disease (GvHD). Has been demonstrated, to our knowledge for the first time, that aHSCT in pediatric patients is associated to a profound modification of the GM ecosystem with a disruption of its mutualistic asset. aGvHD and non-aGvHD subjects showed differences in the process of GM recovery, in members abundance of the phylum Bacteroidetes, and in propionate fecal concentration; the latter are higher in the pre-HSCT composition of non-GvHD subjects than GvHD ones. Short-chain fatty acids (SCFAs), such as acetate, butyrate and propionate, are end-products of microbial fermentation of macronutrients and distribute systemically from the gut to blood. For this reason, has been studied their effect in vitro on human DCs, the key regulators of our immune system and the main player of aGvHD onset. Has been observed that propionate and, particularly, butyrate show a strong and direct immunomodulatory activity on DCs reducing inflammatory markers such as chemokines and interleukins. This study, with the needed caution, suggests that the pre-existing GM structure can be protective against aGvHD onset, exerting its protective role through SCFAs. They, indeed, may regulate cell traffic within secondary lymphoid tissues, influence T cell development during antigen recognition, and, thus, directly shape the immune system.

Reduced muscle mass and bone size in pediatric patients with inflammatory bowel disease

BACKGROUND: Decreased bone mineral density has been reported in children with inflammatory bowel disease (IBD). We used peripheral quantitative computed tomography (pQCT) to assess bone mineralization, geometry, and muscle cross-sectional area (CSA) in pediatric IBD. METHODS: In a cross-sectional study, pQCT of the forearm was applied in 143 IBD patients (mean age 13.9 +/- 3.5 years); 29% were newly diagnosed, 98 had Crohn's disease, and 45 had ulcerative colitis. Auxological data, cumulative glucocorticoid dose, disease activity indices, laboratory markers for inflammation, and bone metabolism were related to the results of pQCT. RESULTS: Patients were compromised in height (-0.82 +/- 1.1 SD), weight (-0.77 +/- 1.0 SD), muscle mass (-1.12 +/- 1.0 SD), and total bone cross-sectional area (-0.79 +/- 1.0 SD) compared to age- and sex-matched healthy controls (z-scores). In newly diagnosed patients, the ratio of bone mineral mass per muscle CSA was higher than in those with longer disease duration (1.00 versus 0.30, P = 0.007). Serum albumin level and disease activity correlated with muscle mass, accounting for 41.0% of variability in muscle mass (P < 0.01). The trabecular bone mineral density z-score was on average at the lower normal level (-0.40 +/- 1.3 SD, P < 0.05). CONCLUSIONS: Reduced bone geometry was explained only in part by reduced height. Bone disease in children with IBD seems to be secondary to muscle wasting, which is already present at diagnosis. With longer disease duration, bone adapts to the lower muscle CSA. Serum albumin concentration is a good marker for muscle wasting and abnormal bone development.

Autotransfusion system or integrated automatic suction device in minimized extracorporeal circulation: influence on coagulation and inflammatory response

To measure surrogate markers of coagulation activation as well as of the systemic inflammatory response in patients undergoing primary elective coronary artery bypass grafting (CABG) using either the so-called Smart suction device or a continuous autotransfusion system (C.A.T.S.®).

Assessment of endothelium and inflammatory response at the onset of reperfusion injury in hand surgery

Background Activation of the endothelium, complement activation and generation of cytokines are known events during ischemia-reperfusion (I/R) that mediate tissue injury. Our aim was to elucidate their respective participation at the onset of the reperfusion phase. Tourniquet application in hand surgery causes short-term ischemia, followed by reperfusion and was therefore used as the model in this study. Methods Ten patients were included in the study after obtaining informed consent. A tourniquet was placed on the upper arm and inflated to 250 mmHg for 116 ± 16 min, during which the surgery was performed. Venous blood and tissue samples from the surgical area were taken at baseline as well as 0, 2, and 10 min after reperfusion and analyzed for the following parameters: Endothelial integrity and/or activation were analyzed by measuring heparan sulfate and syndecan-1 in serum, and vWF, heparan sulfate proteoglycan as well as CD31on tissue. Complement activation was determined by C3a and C4d levels in plasma, levels of C1-inhibitor in serum, and IgG, IgM, C3b/c, and C4b/c deposition on tissue. Cytokines and growth factors IL-5, IL-6, IL-7, IL-8, IL-10, IL-17, G-CSF, GM-CSF, MCP-1, TNFα, VEGF, and PDGF bb were measured in the serum. Finally, CK-MM levels were determined in plasma as a measure for muscle necrosis. Results Markers for endothelial activation and/or integrity as well as complement activation showed no significant changes until 10 min reperfusion. Among the measured cytokines, IL-6, IL-7, IL-17, TNFα, GM-CSF, VEGF, and PDGF bb were significantly increased at 10 min reperfusion with respect to baseline. CK-MM showed a rise from baseline at the onset of reperfusion (p < 0.001) and dropped again at 2 min (p < 0.01) reperfusion, suggesting ischemic muscle damage. Conclusions In this clinical model of I/R injury no damage to the endothelium, antibody deposition or complement activation were observed during early reperfusion. However, an increase of pro-inflammatory cytokines and growth factors was shown, suggesting a contribution of these molecules in the early stages of I/R injury.

Bacterial and inflammatory behavior of implants in the early healing phase of chronic periodontitis

To assess the pattern of early bacterial colonization at implants and teeth in patients with a history of chronic periodontitis compared with a group of healthy subjects. Furthermore, the presence of host-derived markers at teeth and implants in the two subject groups was determined.

Adipocytokines, hepatic and inflammatory biomarkers and incidence of type 2 diabetes. the CoLaus study

Context There is contradictory information regarding the prognostic importance of adipocytokines, hepatic and inflammatory biomarkers on the incidence of type 2 diabetes. The objective was to assess the prognostic relevance of adipocytokine and inflammatory markers (C-reactive protein – CRP; interleukin-1beta – IL-1β; interleukin-6– IL-6; tumour necrosis factor-α – TNF-α; leptin and adiponectin) and gamma-glutamyl transpeptidase (γGT) on the incidence of type 2 diabetes. Methods Prospective, population-based study including 3,842 non-diabetic participants (43.3% men, age range 35 to 75 years), followed for an average of 5.5 years (2003–2008). The endpoint was the occurrence of type 2 diabetes. Results 208 participants (5.4%, 66 women) developed type 2 diabetes during follow-up. On univariate analysis, participants who developed type 2 diabetes had significantly higher baseline levels of IL-6, CRP, leptin and γGT, and lower levels of adiponectin than participants who remained free of type 2 diabetes. After adjusting for a validated type 2 diabetes risk score, only the associations with adiponectin: Odds Ratio and (95% confidence interval): 0.97 (0.64–1.47), 0.84 (0.55–1.30) and 0.64 (0.40–1.03) for the second, third and forth gender-specific quartiles respectively, remained significant (P-value for trend = 0.05). Adding each marker to a validated type 2 diabetes risk score (including age, family history of type 2 diabetes, height, waist circumference, resting heart rate, presence of hypertension, HDL cholesterol, triglycerides, fasting glucose and serum uric acid) did not improve the area under the ROC or the net reclassification index; similar findings were obtained when the markers were combined, when the markers were used as continuous (log-transformed) variables or when gender-specific quartiles were used. Conclusion Decreased adiponectin levels are associated with an increased risk for incident type 2 diabetes, but they seem to add little information regarding the risk of developing type 2 diabetes to a validated risk score.

Folliculo-stellate cells of "true dendritic" type are involved in the inflammatory microenvironment of tumor immunosurveillance of pituitary adenomas

Folliculo-stellate cells are a nonendocrine, sustentacular-like complementary population of the anterior pituitary. They currently are considered as functionally and phenotypically heterogeneous, with one subpopulation of folliculo-stellate cells possibly representing resident adenohypophyseal macrophages. We took advantage of a limited T-cell mediated inflammatory reaction selectively involving tumor tissue in three cases of pituitary adenoma (2 prolactin cell adenomas, and 1 null cell adenoma) to test the hypothesis whether some folliculo-stellate cells within inflammatory foci would also assume monocytic/dendritic properties. Immunohistochemical double labeling for S-100 protein and the class II major histocompatibility antigen HLA-DR indeed showed several arborized cells to coexpress both epitopes. These were distributed both amidst adenomatous acini and along intratumoral vessels, and were morphologically undistinguishable from conventional folliculo-stellate cells. On the other hand, markers of follicular dendritic cells (CD21) and Langerhans' cells (CD1a) tested negative. Furthermore, no S-100/HLA-DR coexpressing folliculo-stellate cells were seen in either peritumoral parenchyma of the cases in point nor in control pituitary adenomas lacking inflammatory reaction. These findings suggest that a subset of folliculo-stellate cells may be induced by an appropriate local inflammatory microenvironment to assume a dendritic cell-like immunophenotype recognizable by their coexpression of S-100 protein and HLA-DR. By analogy with HLA-DR expressing cells in well-established extrapituitary inflammatory constellations, we speculate that folliculo-stellate cells with such immunophenotype may actually perform professional antigen presentation. A distinctly uncommon finding in pituitary adenomas, lymphocytic infiltrates may therefore be read as a manifestation of tumoral immunosurveillance.

Oxidative stress precedes peak systemic inflammatory response in pediatric patients undergoing cardiopulmonary bypass operation

Oxidative stress seems to contribute to cardiopulmonary bypass (CPB)-related postoperative complications. Pediatric patients are particularly prone to these complications. With this in mind, we measured oxidative stress markers in blood plasma of 20 children undergoing elective heart surgery before, during, and up to 48 h after cessation of CPB, along with inflammatory parameters and full analysis of iron status. Ascorbate levels were decreased by approximately 50% (P < 0.001) at the time of aorta cross-clamp removal (or pump switch-off in 4 patients with partial CPB), and associated with corresponding increases in dehydroascorbate (P < 0.001, r = -0.80) and malondialdehyde (P < 0.01, r = -0.59). In contrast to the immediate oxidative response, peak levels of IL-6 and IL-8 were not observed until 3-12 h after CPB cessation. The early loss of ascorbate correlated with duration of CPB (P < 0.002, r = 0.72), plasma hemoglobin after cross-clamp removal (P < 0.001, r = 0.70), and IL-6 and IL-8 levels at 24 and 48 h after CPB (P < 0.01), but not with postoperative lactate levels, strongly suggesting that hemolysis, and not inflammation or ischemia, was the main cause of early oxidative stress. The correlation of ventilation time with early changes in ascorbate (P < 0.02, r = 0.55), plasma hemoglobin (P < 0.01, r = 0.60), and malondialdehyde (P < 0.02, r = 0.54) suggests that hemolysis-induced oxidative stress may be an underlying cause of CPB-associated pulmonary dysfunction. Optimization of surgical procedures or therapeutic intervention that minimize hemolysis (e.g., off-pump surgery) or the resultant oxidative stress (e.g., antioxidant treatment) should be considered as possible strategies to lower the rate of postoperative complications in pediatric CPB.

Novel genetic risk markers for ulcerative colitis in the IL2/IL21 region are in epistasis with IL23R and suggest a common genetic background for ulcerative colitis and celiac disease

OBJECTIVES: Recently, a genome-wide association study showed that single-nucleotide polymorphisms (SNPs) in the chromosome 4q27 region containing IL2 and IL21 are associated with celiac disease. Given the increased prevalence of inflammatory bowel disease (IBD) among celiac disease patients, we investigated the possible involvement of these SNPs in IBD. METHODS: Five SNPs strongly associated with celiac disease within the KIAA1109/TENR/IL2/IL21 linkage disequilibrium block on chromosome 4q27 and one coding SNP within the IL21 gene were analyzed in a large German IBD cohort. The study population comprised a total of 2,948 Caucasian individuals, including 1,461 IBD patients (ulcerative colitis (UC): n=514, Crohn's disease (CD): n=947) and 1,487 healthy unrelated controls. RESULTS: Three of the five celiac disease risk markers had a protective effect on UC susceptibility, and this effect remained significant after correcting for multiple testing: rs6840978: P=0.0082, P(corr)=0.049, odds ratio (OR) 0.77, 95% confidence interval (CI) 0.63-0.93; rs6822844: P=0.0028, P(corr)=0.017, OR 0.73, 95% CI 0.59-0.90; rs13119723: P=0.0058, P(corr)=0.035, OR 0.75, 95% CI 0.61-0.92. A haplotype consisting of the six SNPs tested was markedly associated with UC susceptibility (P=0.0025, P(corr)=0.015, OR 0.72, 95% CI 0.58-0.89). Moreover, in UC, epistasis was observed between the IL23R SNP rs1004819 and three SNPs in the KIAA1109/TENR/IL2/IL21 block (rs13151961, rs13119723, and rs6822844). CONCLUSIONS: Similar to other autoimmune diseases such as celiac disease, rheumatoid arthritis, type 1 diabetes, Graves' disease, and psoriatic arthritis, genetic variation in the chromosome 4q27 region predisposes to UC, suggesting a common genetic background for these diseases.

1.59 Reduced inflammatory potential of circulating gammadelta T cells in pregnancy induced improvement of Rheumatoid Arthritis

BACKGROUND AND OBJECTIVES During pregnancy, gammadelta T cells expand at the fetomaternal interface where they induce a tolerogenic milieu. Patients with rheumatoid arthritis (RA) experience a spontaneous improvement of their disease during pregnancy and a postpartum aggravation. By contrast, pregnant patients with ankylosing spondylitis (AS) often experience persistent active disease. We hypothesised that the pregnancy related modulation of disease activity in RA patients versus AS patients is associated with numerical and functional changes of circulating gammadelta T cells. MATERIAL AND METHODS The frequency of surface markers and the intracellular cytokine profile of freshly isolated gammadelta T cells from RA (n = 54) and AS (n = 26) patients and healthy controls (n = 40) were analysed at each trimester during pregnancy and 6-8 weeks postpartum by flow cytometry. RESULTS Very discrete changes of Vdelta1 or Vdelta2 frequency were seen during pregnancy and postpartum in healthy controls and AS patients. In RA, however, the frequency of Vdelta2 cells decreased in the third trimester when disease activity was low. Low percentages of Vdelta 2 cells were also found in non-pregnant RA patients with active arthritis, yet only pregnant RA patients showed reduced percentages of Vdelta2 cells positive for the activation marker CD69 and the intracellular cytokine TNFalpha. Similarly, Vdelta1 + TNFalpha + cells were lower in pregnant RA patients compared to non-pregnant RA patients. The percentage of Vdelta2 + TNFalpha + cells, Vdelta2+ CD69+ and Vdelta1+ CD69+ cells correlated with disease activity in RA. As for the receptors which modulate cytotoxicity, RA patients showed a rise of the anti-cytotoxic receptor NKG2A on Vdelta1 cells in the 2(nd) trimester and a decrease postpartum. Since the pro-cytotoxic receptor NKG2D remained unchanged, the NKG2D/NKG2A ratio on Vdelta1 cells was reduced in RA patients during pregnancy. In AS patients, persistent disease activity during pregnancy was reflected by an increased frequency of Vdelta2+ CD69+ cells and an unchanged frequency of Vdelta2+ TNFalpha+ cells. In addition, pregnant AS patients showed an increased frequency of Vdelta1+CD161+ cells. CONCLUSIONS Disease amelioration of RA during pregnancy correlates with changes of cell activation, pro-inflammatory cytokines and anti-cytotoxic receptors of gammadelta T cells. By contrast, active disease during pregnancy as found in AS is associated with unchanged inflammatory responses of gammadelta T cells. Since gammadelta T cells remain unchanged in healthy pregnant controls, the modulation of gammadelta T cells in RA rather seems to be an effect of improved disease than of pregnancy itself.

Development of a bead-based multiplex assay for the simultaneous detection of porcine inflammation markers using xMAP technology

Commercially available assays for the simultaneous detection of multiple inflammatory and cardiac markers in porcine blood samples are currently lacking. Therefore, this study was aimed at developing a bead-based, multiplexed flow cytometric assay to simultaneously detect porcine cytokines [interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor alpha], chemokines (IL-8 and monocyte chemotactic protein 1), growth factors [basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and platelet-derived growth factor-bb], and injury markers (cardiac troponin-I) as well as complement activation markers (C5a and sC5b-9). The method was based on the Luminex xMAP technology, resulting in the assembly of a 6- and 11-plex from the respective individual singleplex situation. The assay was evaluated for dynamic range, sensitivity, cross-reactivity, intra-assay and interassay variance, spike recovery, and correlation between multiplex and commercially available enzyme-linked immunosorbent assay as well as the respective singleplex. The limit of detection ranged from 2.5 to 30,000 pg/ml for all analytes (6- and 11-plex assays), except for soluble C5b-9 with a detection range of 2-10,000 ng/ml (11-plex). Typically, very low cross-reactivity (<3% and <1.4% by 11- and 6-plex, respectively) between analytes was found. Intra-assay variances ranged from 4.9 to 7.4% (6-plex) and 5.3 to 12.9% (11-plex). Interassay variances for cytokines were between 8.1 and 28.8% (6-plex) and 10.1 and 26.4% (11-plex). Correlation coefficients with singleplex assays for 6-plex as well as for 11-plex were high, ranging from 0.988 to 0.997 and 0.913 to 0.999, respectively. In this study, a bead-based porcine 11-plex and 6-plex assay with a good assay sensitivity, broad dynamic range, and low intra-assay variance and cross-reactivity was established. These assays therefore represent a new, useful tool for the analysis of samples generated from experiments with pigs.

Comparison of the toxicity of diesel exhaust produced by bio- and fossil diesel combustion in human lung cells in vitro

Alternative fuels are increasingly combusted in diesel- and gasoline engines and the contribution of such exhausts to the overall air pollution is on the rise. Recent findings on the possible adverse effects of biodiesel exhaust are contradictive, at least partly resulting from the various fuel qualities, engine types and different operation conditions that were tested. However, most of the studies are biased by undesired interactions between the exhaust samples and biological culture media. We here report how complete, freshly produced exhausts from fossil diesel (B0), from a blend of 20% rapeseed-methyl ester (RME) and 80% fossil diesel (B20) and from pure rapeseed methyl ester (B100) affect a complex 3D cellular model of the human airway epithelium in vitro by exposing the cells at the air–liquid interface. The induction of pro-apoptotic and necrotic cell death, cellular morphology, oxidative stress, and pro-inflammatory responses were assessed. Compared to B0 exhaust, B20 exhaust decreased oxidative stress and pro-inflammatory responses, whereas B100 exhaust, depending on exposure duration, decreased oxidative stress but increased pro-inflammatory responses. The effects are only very weak and given the compared to fossil diesel higher ecological sustainability of biodiesel, it appears that – at least RME – can be considered a valuable alternative to pure fossil diesel.