Effects of Dietary Omega-3 Enrichment on the Chemical Composition and the Pathogenic Microbiota of Ovine Milk.

The demand for ovine milk and ovine dairy products is constantly increasing due to their exceptional sensorial characteristics and their health benefits for consumers. However, dairy fat content and composition are of particular concern for consumers as well as the medical community, as there are risk factors for coronary disease, diabetes mellitus, cancer, and other serious diseases. For this reason, attempts have been made to control/regulate the fat composition of ovine milk by modifying sheep dietary intake of polyunsaturated fatty acids. In this experimental trial, a group of sheep were fed for 30 days a diet enriched in flaxseeds and lupines, feed ingredients rich in omega-3 fatty acids, aiming to investigate the effects on fat composition and the microbiota of ovine milk. Chemical analysis of the collected milk showed that the omega-3 and omega-6 content was increased. On the opposite, the atherogenic and thrombogenic indexes decreased. Of importance was the semi-protective effect on the udder by the increased omega-3 dietary intake, as depicted by its impact on the biodiversity of the pathogenic microbiota. These findings suggest that ovine milk could be modified under specific conditions to be more appropriate for the consumption by people belonging to high-risk groups for various diseases.

Identification by MALDI-TOF MS and Antibiotic Resistance of Riemerella anatipestifer, Isolated from a Clinical Case in Commercial Broiler Chickens.

The Gram-negative bacterium Riemerella anatipestifer (RA) is known to cause clinical disease with severe economic impacts primarily in ducks and less frequently in geese and turkeys. RA was isolated and identified in broiler chickens, from a clinical case in a commercial broiler farm located in the southwest mainland of Greece. The morbidity and the mortality in the broiler house were estimated at 10% and 5% respectively. The observed clinical signs appeared at the age of 30 to 42 days with respiratory distress (dyspnea), white fluid feces and stunting. Post-mortem examinations displayed serositis, pericarditis, perihepatitis and airsacculitis. Edematous swelling around the tibio-tarsal joints was observed in some birds. Tissue samples from lesions were streaked on selective media. Three bacterial isolates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Moreover, an antibiogram analysis was performed for the three RA strains, using a pattern of 16 common antibiotics to advocate the most effective drugs for a proper treatment. All the RA isolates were sensitive to ceftiofur, sulphamethoxazole-trimethoprim and amoxicillin, whereas all were resistant to gentamicin, tylosin, tetracyclin, colistin sulphate, spectinomycin, lincomycin and oxytetracycline.

The regulation of serum resistin levels in metabolically healthy and unhealthy obese individuals.

PURPOSE: We have recently demonstrated that absolute counts of circulating proinflammatory monocytes were lower in obese patients without metabolic syndrome (MS) (metabolically healthy obese, MHO) compared with those with MS (metabolically unhealthy obese, MUO), but higher compared with healthy lean controls (MHL). We hypothesized that circulating resistin, a cytokine secreted by white blood cells (WBC), is involved in obesity-related low-grade inflammation. The aim of this study was to (a) determine serum resistin levels among MUO and MHO subjects and (b) investigate the role of circulating WBC subsets as potential determinants of resistin. METHODS: Study participants were 58 obese (33 MUO, 25 MHO) and 25 MHL individuals. Serum levels of resistin, high-sensitivity C-reactive protein (hsCRP), and absolute counts of circulating WBC subpopulations were determined. Comparisons were sex- and age-adjusted. RESULTS: Serum resistin levels in MHL were lower compared with those of obese (p = 0.041), but similar to those of MHO (p = 0.856) individuals. Both resistin (p = 0.005) and absolute neutrophil count (NeuA) (p = 0.025) were higher in MUO compared with MHO. The difference in resistin levels between obese and MHL individuals disappeared after adjustment for NeuA. Resistin correlated positively with absolute total monocyte count (p = 0.037) in MHL and with body mass index (BMI) (p = 0.023), hsCRP (p = 0.022), and NeuA (p = 0.044) in obese subjects. Resistin association with ΒΜΙ disappeared after adjustment for hsCRP, while association with hsCRP disappeared after further adjustment for NeuA. CONCLUSION: Circulating resistin was higher in MUO compared with MHO. The increased secretion of resistin by the greater number of neutrophils in the former may have contributed to this regulation.

A modified flow cytometry method for objective estimation of human CD4+ regulatory T cells (CD4+ Tregs) in peripheral blood, via CD4/CD25/CD45RO/FoxP3 labeling.

BACKGROUND: Several methods exist for flow-cytometric estimation of human peripheral blood CD4+ T regulatory cells (CD4+ Tregs). METHODS: We report our experience with the estimation of human CD4+ Tregs via three different characterizations using flow cytometry (CD25high FoxP3+ , CD25high CD127low/- FoxP3+ , and CD4+ CD25high/int CD45ROFoxP3+ ) in normal subjects. We have used these methods on the control populations from two studies (32 and 36 subjects, respectively), the latter two methods retrospectively on the subjects of the first study. The six CD4+ T cell fractions obtained by the third method were differentially colored to ascertain the distribution of these cell fractions in the CD25/FoxP3, CD45RO/FoxP3, and CD25/CD127 dot plots from CD4/CD25/CD45RO/FoxP3 and CD4/CD25/CD45RO/CD127 panels. RESULTS: Each approach gives significantly different estimates of Tregs (expressed as percentage of CD4+ T cells), with the second almost invariably yielding higher percentages than the other two. Only the third approach can distinguish among effector and naïve Tregs and FoxP3+ non-Tregs. Analysis of CD25/CD127 dot plots reveals that Treg delineation via the widely used definition of CD4+ CD25high CD127low/- cells unavoidably yields a mixture of nearly all effector and most of naïve Tregs, as well as FoxP3+ non-Tregs plus other cells. Delineation of effector/naïve Tregs and FoxP3+ non-Tregs is possible via CD45RO/CD25 dot plots but not by CD45RO/FoxP3 counterparts (as done previously) because of overlapping FoxP3 intensities among Tregs and non-Tregs. CONCLUSION: Our comparison shows that CD4/CD25/CD45RO/FoxP3 panels are an objective means of estimating effector and naïve Tregs via colored dot plots, aiding thus in Treg delineation in health and detecting aberrations in disease.

Metabolically Healthy Obesity Is Characterized by a Proinflammatory Phenotype of Circulating Monocyte Subsets.

Background: Obesity is associated with macrophage infiltration in adipose tissue that induces insulin resistance and contributes to the development of metabolic syndrome (MS). The aim of this study was to investigate whether circulating monocyte subsets (macrophage precursors) differ among obese subjects with MS [metabolically unhealthy obese (MUO)], obese subjects without MS [metabolically healthy obese (MHO)], and metabolically healthy lean (MHL) individuals. Methods: Fifty-eight obese (33 MUO, 25 MHO) and 25 MHL individuals participated in the study. Absolute blood counts of classical (Mon1A), intermediate (Mon2A), and nonclassical (Mon3A) monocyte subsets were measured by flow cytometry. Results: Increased proinflammatory monocyte counts (Mon2A, Mon3A) were observed in obese compared with MHL individuals (P = 0.001 and P = 0.017 respectively). Mon2A count in MHO was lower compared with that in MUO subjects (P = 0.036) but higher compared with MHL controls (P = 0.032). Mon2A was positively associated with serum triglyceride levels (r = 0.328, P = 0.023) and mean blood pressure (BP) (r = 0.457, P = 0.001) in obese subjects. Among MS components, only the presence of elevated BP (≥130/85 mmHg) was independently associated with increased Mon2A in obese subjects (P < 0.001). Conclusions: Absolute counts of proinflammatory monocytes were lower in metabolically healthy compared with MUO individuals, but higher compared with healthy lean controls. The presence of low-grade inflammation suggests that "metabolically healthy" obesity is not a benign condition. ClinicalTrials.gov identifier: NCT03241394.

Effects of Oregano, Attapulgite, Benzoic Acid and their Blend on Chicken Performance, Intestinal Microbiology and Intestinal Morphology.

The aim of the present study was to investigate the effects of oregano, attapulgite, benzoic acid and their combination on broiler performance, microflora composition of jejunum and cecum, intestinal architecture and breast and thigh meat composition. A total of 400 one-day-old broiler chicks were used in a 42-day trial. They were randomly distributed into five treatments with four replicates of twenty chickens per pen: Control group; Attapulgite group; Oregano essential oil group; Benzoic acid group; Mixed group. At the end of the trial, total counts of bacteria, Enterobacteriaceae, Lactobacilli, and Clostridium perfringens were enumerated by real time PCR at both jejunum and cecum. Intestinal morphology was carried out in duodenum, jejunum and ileum, for villus height and crypt depth. Cell proliferation was also evaluated in the small intestine and the cecum. The results showed that oregano and benzoic acid improved some growth performance parameters. The combined use of the examined substances increased enterobacteria counts in the jejunum, and cell proliferation in the duodenum and the jejunum. Benzoic acid improved intestinal wall morphology in the ileum. In conclusion, the combined dietary supplementation with oregano, attapulgite and benzoic acid can be a novel tool to beneficially modulate broiler chickens performance.

Platelet activation after endovascular repair of abdominal aortic aneurysm.

AIM: To investigate the effect of endovascular aneurysm repair (EVAR) on platelet (PLT) function and reveal possible associated factors. METHODS: Fifty consecutive patients were included. PLT count and activation (CD62P-CD36), white blood cell (WBC) count, and high sensitivity C-reactive protein (hs-CRP) were measured preoperatively, at the first and third postoperative day. RESULTS: EVAR elicited a significant reduction in PLT count from baseline to first day after EVAR (p < 0.001), while no significant difference was noted between the first and third day. Furthermore, CD62P expression was markedly elevated at the first day after EVAR (median % positive PLTs from 13.7 at baseline to 22.1, p = 0.05), but returned to baseline levels by the third day. Maximum abdominal aortic aneurysm diameter was the only factor that significantly affected the CD62P values (p = 0.005). Postoperative CD36 values were significantly correlated with total aneurysm volume (p = 0.05) and were higher in endografts made from polyester (p = 0.01). There were no correlation between PLT activation and hs-CRP, WBC, maximum temperature, and 30-day morbidity. CONCLUSION: EVAR has elucidated a significant reduction in PLT count and increase in PLT activation at the immediate postoperative period. The type of the endograft material and the aneurysm maximum diameter and volume appear to play an important role in PLT activation.

Expression of heat shock proteins in brain tumors.

AIM: Heat shock proteins (HSP) are an evolutionary conserved family of proteins that serve as molecular chaperones, preventing the formation of nonspecific protein aggregates and assisting proteins in the acquisition of their native structures. Furthermore, HSPs have anti-apoptotic properties and have been found to be elevated in many human cancers; their overexpression has been associated with poor survival and response to therapy. In the present study we assessed the HSP expression in brain tumors. MATERIAL AND METHODS: Simultaneous detection of HSP27, HSP40, HSP60, HSP70, HSP90a, total Akt and phospho- Akt in 19 brain tumor specimens was performed using the multiplex bead array assay. RESULTS: There was expression of HSP27 (pSer82), HSP27 (pSer15), HSP40, HSP60, HSP70, HSP90a, total-Akt and phospho- Akt in both gliomas and meningiomas. Significantly higher levels of HSP70 and a trend towards higher levels of HSP40 were found in meningiomas compared to gliomas. There was a significant correlation between HSP27 (pSer82) and HSP27 (pSer15) expression and between HSP90a and both total-AKT and phospho- AKT. A significant correlation between HSP27 and total-AKT was observed. CONCLUSION: Since HSPss are an attractive target for anticancer therapy, further studies are needed in order to better assess their relationship with tumor aggressiveness and patient prognosis.

Changes of serum adhesion molecules and cytokines in post-ERCP pancreatitis: adhesion molecules and cytokines in acute pancreatitis.

OBJECTIVES: To assess the early changes of soluble IFN-γ, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, TNF-α, TNF-ß, IL-17A, IL-22, soluble (s) P-Selectin, sE-Selectin and sICAM-1 in post-ERCP pancreatitis (PEP). METHODS: Single center, prospective study of 318 ERCP procedures. Serum samples were acquired from all patients prior to ERCP, 6 hours and 24 hours after the procedure. For every PEP case, another patient was chosen as a control, matched for gender, age and time period in which ERCP took place. RESULTS: Totally, 28 cases and 28 controls were studied. Except for significantly higher IL-1b levels in cases at baseline, no significant differences were observed between cases and controls after Bonferroni corrections. An increase in IL-6 was noted between baseline and 6 h in cases alone (p=0.016). There was a significant fall in sP-selectin levels at 6 and 24 hours compared to baseline in all patients (corrected p=0.008 and 0.016 for cases and 0.016 and 0.048 for controls respectively). An increase of sE-selectin in cases was observed between 6 and 24 hours post-ERCP (corrected p=0.03). CONCLUSIONS: Soluble forms of cytokines and adhesion molecules studied seem not to play a major role in PEP.

Expression of heat shock proteins in medulloblastoma.

OBJECT: Medulloblastoma (MB) is the most common malignant brain tumor in children. Heat shock proteins (HSPs) comprise a superfamily of proteins that serve as molecular chaperones and are overexpressed in a wide range of human cancers. The purpose of the present study was to investigate the expression of HSP27 (pSer(82)), HSP27 (pSer(15)), HSP40, HSP60, HSP70, HSP90-α, Akt, and phospho-Akt by multiplex bead array assay of MBs. The results of HSP and Akt expression were correlated with MB subtype; immunohistochemical expression of Ki-67 index, bcl-2, and p53; and patients' prognosis. METHODS: The authors retrospectively evaluated 25 children with MB who underwent surgery. Immunohistochemical analysis of Ki-67, p53, and bcl-2 expression was performed in all cases. By using multiplex bead array assay, a simultaneous detection of HSP27 (pSer(82)), HSP27 (pSer(15)), HSP40, HSP60, HSP70, HSP90-α, Akt, and phospho-Akt was performed. RESULTS: Medulloblastoma with extensive nodularity had significantly lower HSP27 (pSer(15)) expression (p = 0.039) but significantly higher HSP60 expression (p = 0.021) than classic MB. Large-cell MB had significantly higher HSP70 expression (p = 0.028) than classic MB. No significant difference was found between HSP27 (pSer(82)), HSP40, HSP90-α, Akt, or phospho-Akt expression and MB subtype. Large-cell MBs had significantly higher Ki-67 index compared with classic MBs (p = 0.033). When analyzing all MBs, there was a significant negative correlation between HSP27 (pSer(15)) and Ki-67 index (r = -0.475, p = 0.016); a significant positive correlation between HSP70 expression and Ki-67 index (r = 0.407, p = 0.043); and a significant positive correlation between HSP70 expression and bcl-2 index (r = 0.491, p = 0.023). Patients with large-cell MB had a worse survival than those with classic MB, but the difference did not reach statistical significance (p = 0.076). CONCLUSIONS: A substantial expression of several HSPs in MB was observed. Given that HSPs represent an attractive strategy for anticancer therapy, further studies, involving larger series of patients, are obviously necessary to clarify the relationship of HSPs with tumor aggressiveness and prognosis.

DNA content is associated with malignancy of intracranial neoplasms.

OBJECTIVE: Flow cytometry has been applied to analyze the DNA-content distribution of tumors, in order to relate this to clinical and biological parameters of tumor behavior. Herewith, we investigated the value of cell cycle analysis in the characterization of intracranial lesions and its possible prognostic role. METHODS: DNA analysis was performed in tumor samples that were taken during surgery over a five year period. Diagnosed tumors were graded according to the World Health Organization 2007 classification scheme. RESULTS: Fifty-six patients were included in the study. There was a significant difference in G0/G1 phase and S-phase between low-grade and high-grade gliomas. There were 12 (57%) diploid and 9 (43%) aneuploid tumors. All aneuploid tumors were glioblastomas. Patients with G0/G1 value ≤ 69% and S phase value greater than 6% were associated with worse survival. As regards meningiomas, there was a significant difference in G0/G1 phase, S phase and mitoses fraction between benign and both atypical and anaplastic meningiomas. Aneuploidy was observed in the anaplastic tumors and in 2/4 atypical meningiomas. CONCLUSION: The results of the present study, showed that cell cycle analysis could differentiate low from high grade gliomas and benign from atypical/anaplastic meningiomas. Furthermore, a prognostic significance was found in glioma patients. The role of cell cycle analysis in brain tumors thus warrants further investigation.

Circulating progenitor cells: a comparison of patients with glioblastoma or meningioma.

Blood circulating endothelial cells and circulating hematopoietic progenitor cells (CPCs) are two cell populations that are thought to play important role in angiogenesis. In the present study, we investigated the role of CPCs in patients with brain tumors. We prospectively studied 19 brain tumor patients. Ten healthy individuals were used as controls. Variables that were analyzed included age, sex, Ki-67 index, symptom duration, tumor location, tumor size and preoperative Karnofsky performance status score (KPS). CPCs were determined as CD45(dim)/CD34+/CD133+ in the peripheral blood. Twelve patients had glioblastoma (GBM), 1 patient had a grade II glioma and 6 patients had meningioma. Brain tumor patients had significantly higher CPC levels compared to healthy volunteers. Patients with gliomas had significantly higher CPC levels than patients with meningiomas. In GBM patients no correlation was found between CPC levels and sex, age, Ki-67 index, tumor location, size and KPS. Patients with CPC levels lower than 1,743 cells/ml had a higher progression-free survival but the difference was not statistically significant. Glioma patients had higher CPC levels compared to patients with meningiomas. Larger studies are obviously needed to verify the role of CPC levels in patients with brain tumors.

Asymptomatic T-cell large granular lymphocyte leukemia with an unusual immunophenotype.

T-cell large granular lymphocyte (T-LGL) leukemia represents a clonal proliferation of cytotoxic T-cells which etiology has not been entirely elucidated. However, CD4(+), CD4(-), CD8(-), CD4(+), CD8(+) cases have been described. The disease is usually characterized by cytopenias and a modest lymphocytosis. The majority of patients with T-LGL leukemia remains asymptomatic for a long period and will require treatment later during the course of their disease. Hereby we describe a case of T-LGL leukemia diagnosed by flow cytometry, which presented indolent course and required no treatment so far.