Methods in isolation and characterization of bovine monocytes and macrophages.

Monocytes and macrophages belong to the mononuclear phagocyte system and play important roles in both physiological and pathological processes. The cells belonging to the monocyte/macrophage system are structurally and functionally heterogeneous. Several subsets of monocytes have been previously identified in mammalian blood, generating different subpopulations of macrophages in tissues. Although their distribution and phenotype are similar to their human counterpart, bovine monocytes and macrophages feature differences in both functions and purification procedures. The specific roles that monocytes and macrophages fulfil in several important diseases of bovine species, including among the others tuberculosis and paratuberculosis, brucellosis or the disease related to peripartum, remain still partially elusive. The purpose of this review is to discuss the current knowledge of bovine monocytes and macrophages. We will describe methods for their purification and characterization of their major functions, including chemotaxis, phagocytosis and killing, oxidative burst, apoptosis and necrosis. An overview of the flow cytometry and morphological procedures, including cytology, histology and immunohistochemistry, that are currently utilized to describe monocyte and macrophage main populations and functions is presented as well.

The ACACA gene is a potential candidate gene for fat content in sheep milk.

No major gene has yet been reported in sheep that explains the variation of milk fat content. The coding region of the acetyl-CoA carboxylase alpha (ACACA) gene, which plays an important role in de novo fatty acid synthesis, had been investigated, but no non-synonymous mutations have been reported. In this study, the genomic regions encoding the three promoters of the ACACA gene were directly sequenced in 264 sheep of three different breeds, and 10 SNPs were identified. Allele frequencies of most SNPs significantly differed (P = 0.05-0.0001) between breeds. The SNPs that potentially altered either gene regulatory elements or putative binding sites of transcription factors were made evident through in silico analysis. The association analysis with milk traits, performed for one SNP of PIII (GenBank AJ292286, g.1330G>T), showed a significant allelic substitution effect (+0.33%, P < 0.0001 and +0.35%, P < 0.01) in the Altamurana and Gentile breeds respectively. Because this SNP was located in the binding site of the paired box protein transcription factors, which was shown to function as an efficient promoter element, and because PIII transcripts are expressed in the mammary gland, the SNP in PIII of the ACACA gene might affect the variation of fat content in sheep milk.

Analysis of SLC11A1 gene expression in healthy water buffalo (Bubalus bubalis) blood cells using qPCR.

SLC11A1 (solute carrier family 11 member 1 protein) gene influences the initial phase of bacterial cellular infections through macrophage activation. Recent literature on buffalo has attempted to associate the genotype of the polymorphic microsatellite located in the 3'untranslated region (3'UTR) of the gene, with either susceptibility to brucellosis or with improved macrophage function. Carriers of the (GT)16 allele have been reported to be resistant to brucellosis. In this study we analyzed the steady-state level of SLC11A1 expression in a serologically negative herd of 26 animals differing by the number of (GT)n microsatellite repeats by using a reverse transcriptase quantitative real-time polymerase chain reaction approach. We evaluated five different reference genes, which had not been reported previously, for use in gene expression experiments in buffalo blood. However, we did not find any significant difference between buffalo carriers of the different microsatellite alleles, with respect to SLC11A1 expression in whole blood or in blood fractions [peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes/granulocytes (PMN/G)]. Conversely, there was a difference between the blood fractions in their SLC11A1 expression levels, with the PMN/G fraction having a higher expression level than the PBMC fraction (P < 0.015).

Gene expression of pregnancy-associated glycoproteins-1 (PAG-1), interferon-tau (IFNt) and interferon stimulated genes (ISGs) as diagnostic and prognostic markers of maternal-fetal cellular interaction in buffalo cows.

The aim of this study was to determine the presence of Pregnancy-associated glycoprotein -1 (PAG-1) mRNA expression in the maternal circulation of pregnant buffaloes during the early stage of pregnancy. Contemporaneously, the mRNA expression levels of Interferon-tau (IFNt) and some Interferon stimulated genes (ISGs) (interferon stimulated gene 15 ubiquitin-like modifier interferon, ISG15; Mixoviruses resistance 1 and 2, MX1 and MX2; 2',5'-oligoadenylate synthase 1,OAS1) were evaluated in order to expand our knowledge of the molecular processes involved in the early stages of pregnancy and to identify potential biomarkers of maternal-fetal cellular interaction in buffalo. The study was conducted on 38 synchronized and artificially inseminated buffalo cows (d 0), divided ex post into 3 groups: Pregnant (n = 17), Non-pregnant (n = 15) and Embryo mortality (n = 6). Blood samples were collected on d 14, 19, 28 and 40 after artificial insemination (AI) for peripheral blood mononuclear cells (PBMCs) isolation. Expression levels of mRNA of PAG-1, IFNt, ISG15. MX1, MX2 and OAS1 were measured using RT-qPCR. No significant changes were observed in IFNt and PAG gene expressions between groups, while significant differences (p < 0.001) were found for ISG15, MX1, MX2, and OAS1. Pairwise comparisons revealed that the differences between groups occurred on days 19 and 28 post-AI. ISG15 proved to have the best diagnostic performance for distinguishing between pregnant animals and animals that experienced embryo mortality with the ROC analysis. According to the results of the univariate analyses, day 19 was identified as the most indicative to discriminate between groups while the most reliable genes for this differentiation were ISG15, MX1 and MX2. MX2 proved to be the best gene for discriminating pregnant buffaloes using the discriminant analysis, while MX1 was the gene that best predicted embryo mortality. Our results showed that among PAG-1, IFNt and ISGs expression as diagnostic and prognostic markers of maternal-fetal cellular interaction in buffalo cows, ISGs proved to be the best peripheral biomarkers for predicting pregnancy and embryonic mortality during the peri-implantation period. These insights into the mechanisms behind maternal-fetal interaction and the development of a method for the early detection of embryo distress may enable us to implement effective strategies to support embryo survival.

Skyrmion tubes in achiral nematic liquid crystals.

We analyze the interaction with uniform external fields of nematic liquid crystals within a recent generalized free energy posited by Virga and falling in the class of quartic functionals in the spatial gradients of the nematic director. We review some known interesting solutions, i.e., uniform heliconical structures, which correspond to the so-called twist-bend nematic phase and we also study the transition between this phase and the standard uniform nematic one. The twist-bend phase is further reproduced by three-dimensional simulations. Moreover, we find liquid crystal configurations, which closely resemble some novel, experimentally detected, structures called Skyrmion tubes. Skyrmion tubes are characterized by a localized cylindrically symmetric pattern surrounded by either twist-bend or uniform nematic phase. We study the equilibrium differential equations and find numerical solutions and analytical approximations.

Phenotyping congestion in patients with acutely decompensated heart failure with preserved and reduced ejection fraction: The Decongestion duRing therapY for acute decOmpensated heart failure in HFpEF vs HFrEF- DRY-OFF study.

AIMS: To evaluate pulmonary and intravascular congestion at admission and repeatedly during hospitalization for acute decompensated heart failure (ADHF) in HFrEF and HFpEF patients using lung (LUS) and inferior vena cava (IVC) ultrasound. METHODS AND RESULTS: Three-hundred-fourteen patients (82±9 years; HFpEF =172; HFrEF=142) admitted to Internal Medicine wards for ADHF were enrolled in a multi-center prospective study. At admission HFrEF presented higher indexes of pulmonary and intravascular congestion (LUS-score: 0.9 ±â€¯0.4 vs 0.7 ±â€¯0.4; p<0.01; IVC end-expiratory diameter: 21.6 ±â€¯5.1 mm vs 20±5.5 mm, p<0.01; IVC collapsibility index 24.4 ±â€¯17.4% vs 30.9 ±â€¯21.1% p<0.01) and higher Nt-proBNP values (8010 vs 3900 ng/l; p<0.001). At discharge, HFrEF still presented higher B-scores (0.4 ±â€¯4 vs 0.3 ±â€¯0.4; p = 0.023), while intravascular congestion improved to a greater extent, thus IVC measurements were similar in the two groups. No differences in diuretic doses, urine output, hemoconcentration, worsening renal function were found. At 90-days follow up HF readmission/death did not differ in HFpEF and HFrEF (28% vs 31%, p = 0,48). Residual congestion was associated with HF readmission/death considering the whole population; while intravascular congestion predicted readmission/death in the HFrEF, no association between sonographic indexes and the outcome was found in HFpEF. CONCLUSIONS: Serial assessment of pulmonary and intravascular congestion revealed a higher burden of fluid overload in HFrEF and, conversely, a greater reduction in intravascular venous congestion with diuretic treatment. Although other factors beyond EF could play a role in congestion/decongestion patterns, our data may be relevant for further phenotyping HF patients, considering the importance of decongestion optimization in the clinical approach.

Nonuniform localized distortions in generalized elasticity for liquid crystals.

We analyze a recent generalized free-energy for liquid crystals posited by Virga and falling in the class of quartic functionals in the spatial gradients of the nematic director. We review some known interesting solutions, i.e., uniform heliconical structures, and we find new liquid crystal configurations, which closely resemble some novel, experimentally detected, structures called Skyrmion tubes. These new configurations are characterized by a localized pattern given by the variation of the conical angle. We study the equilibrium differential equations and find numerical solutions and analytical approximations.

Ovine acyl CoA:diacylglycerol acyltransferase 1- molecular characterization, polymorphisms and association with milk traits.

The objective of this work was to characterize the complete coding region of the ovine acylCoA:diacylglycerol acyltransferase 1 (DGAT1) gene of three Italian sheep breeds: Sarda, Altamurana and Gentile di Puglia. Characterization was accomplished by direct sequencing of 8676 bp of the relevant DNA, including introns and partial 5' and 3' untranslated regions (UTRs). We detected five novel SNPs; one SNP (g.5553C>T) is located in intron 2, has similar frequencies in the three breeds and showed a negative association with milk fat content. More interesting is an SNP in the 5' UTR (g.127C>A), the occurrence of which is rare in the higher milk-fat breeds (Altamurana and Gentile di Puglia); it is located in the core sequence of Sp1, a putative binding site of transcription factors. This SNP showed a significant negative association with milk fat content in the Sarda sheep. Because DGAT1 plays a fundamental role in triacylglycerol synthesis, the novel detected SNP in the 5' UTR of the DGAT1 gene might explain, at least partially, the variation of fat content in the milk of Sarda sheep.

Helicoids in chiral liquid crystals under external fields.

Cholesteric liquid crystals, subject to externally applied magnetic fields and confined between two parallel planar surfaces with strong homeotropic anchoring conditions, are found to undergo transitions to different types of helicoidal configurations with disclinations. Analytical and numerical studies are performed in order to characterize their properties. In particular, we produce a phase diagram for the transitions from the nematic state to the helicoidal phases in terms of the molecular chirality and the strength of the applied magnetic field.

Association between peripheral arterial disease and cardiovascular risk factors: role of ultrasonography versus ankle-brachial index.

OBJECTIVE: Most studies on atherosclerotic processes include peripheral arterial disease diagnosis only if patients report symptoms suggestive of peripheral arterial disease and/or an instrumental demonstration of lower limbs perfusion deficit is provided, rather than the sole presence of atherosclerotic lesions localized at lower limbs, this attitude leading to ignore early stages of the disease. To overcome these limitations, we have proposed a new ultrasonographic semiquantitative score to better identify all disease stages. The aim of this study is to compare ultrasonography versus ankle-brachial index in the association between peripheral arterial disease and cardiovascular risk factors. PATIENTS AND METHODS: This cross-sectional observational study included subjects undergoing lower limbs evaluation through ultrasonography and ankle-brachial index determination because of symptoms suggestive of peripheral arterial disease or presence of known cardiovascular risk factors. Associations between ultrasonography and ankle-brachial index with cardiovascular risk factors were assessed by first fitting logistic regression models and then comparing the respective areas under the Receiver Operating Characteristic and 95% confidence intervals. RESULTS: The areas under the Receiver Operating Characteristic for each cardiovascular risk factors were consistently larger in magnitude for ultrasonography compared with ankle-brachial index, this comparison being statistically significant for age, male gender, smoking status, hypertension, diabetes mellitus and previous cardiovascular events. CONCLUSIONS: Our study demonstrates that ultrasonography is a better method to screen peripheral arterial disease respect to ankle-brachial index in order to identify all disease stages. These findings are useful in particular when including peripheral arterial disease as organ damage marker in cardiovascular risk stratification.

Short communication: Effect of stearoyl-coenzyme A desaturase polymorphism on fatty acid composition of milk.

The effect of the stearoyl-CoA desaturase (SCD) gene on milk fatty acid composition was tested. Cows of 3 breeds of northern Italy, Piedmontese, Valdostana, and Jersey, were genotyped at exon 5 of the SCD gene. This has been suggested as a primary candidate gene to change the proportion of saturated vs. unsaturated fatty acids in milk, wherein a single nucleotide polymorphism (C/T) gives rise to a different AA codon. It was possible to ascribe a reduced desaturase activity to the T allele only in the case of caproleic and myristoleic fatty acids. In contrast with the findings of SCD effects on carcass fat, it was not possible to confirm the higher desaturation activity of this single nucleotide polymorphism on long-chain fatty acids, due to the different pathways that originate milk fatty acids of different carbon length; long-chain fatty acids are highly influenced by the complex metabolic events that affect the ingested nutrients during their transfer to milk fat.

Atherosclerosis and cardiovascular involvement in celiac disease: the role of autoimmunity and inflammation.

OBJECTIVE: The aim of this review is to explore the evidence about the association among celiac disease (CD), atherosclerosis (AS) and cardiovascular (CV) diseases, and the role of inflammation in this connection. MATERIALS AND METHODS: A systematic literature search was conducted using PubMed, EMBASE, and Cochrane Library for the association among CD, AS and CV diseases. RESULTS: Several studies reported the association of CD with accelerated AS, as evidenced by the alterations of a number of parameters indicative of subclinical AS, as increased carotid artery intima-media thickness, endothelial dysfunction and increased arterial stiffness. In addition, recent evidence reported an increase of CV diseases prevalence in CD patients respect to controls, many of which including ischemic diseases as acute myocardial infarction and angina pectoris, as well as death from ischemic heart disease, and, more rarely, stroke for cerebrovascular involvement. Other not-ischemic CV diseases associated with CD are represented by dilated cardiomyopathy, atrial fibrillation, and myocarditis. CONCLUSIONS: On the basis of the reported association among CD, AS and CV diseases, we suggest to perform a more detailed CV risk assessment in all CD patients than what is currently being achieved in clinical practice, in order to scan and treat modifiable CV risk factors in these patients. In particular, we suggest to resort to instrumental techniques to detect AS in the subclinical stage, in order to prevent AS development and CV diseases in CD patients.

Focus on biological identity of endothelial progenitors cells.

Circulating Endothelial Progenitor Cells (EPCs) were discovered by Asahara et al in 1997 and defined as bone marrow CD34+/KDR+ cells endowed with angiogenic potentialities in vitro and in vivo. The most likely assumption is that EPCs consist of several cell subpopulations with functions targeted at accomplishing the post-natal neovascularization process in a synergic and complementary fashion. Indeed, the subsequent identification of numerous and differentiated hematic populations, characterized by the capacity to develop an endothelial phenotype, has posed a number of questions as to the real identity of EPCs. This concept does not represent a sterile speculation but rather it suggests important implications for the future practice of stem cell therapy. The aim of this report was to explore through a critical analysis the two main experimental methodologies, in vitro culture and flow cytometry, applied to EPCs, followed by a brief revaluation of the endothelial progenitors employing a globally functional approach.

Extent and pattern of regression of left ventricular hypertrophy in patients with small size CarboMedics aortic valves.

OBJECTIVE: To assess the extent and pattern of regression of left ventricular hypertrophy after valve replacement for aortic stenosis, we studied 26 patients receiving either 19 or 21 mm CarboMedics valves (group I, 13 patients) or either 23 or 25 mm CarboMedics valves (group II, 13 patients). The studies were done before the operation and after 3 years, and results were compared with those of 10 control patients. METHODS: Left ventricular end-diastolic and end-systolic diameters and volumes, ejection fraction and fractional shortening, and interventricular septum and posterior wall thickness were measured. The ratio between interventricular septum and posterior wall thickness, the ratio between left ventricular wall thickness and left ventricular chamber radius, and the left ventricular mass were then calculated. RESULTS: At follow-up there was a significant reduction in the left ventricular mass, interventricular septum, and posterior wall thickness for both patient groups (p < 0.01). However, only the posterior wall thickness reached normal values; the interventricular septum and the left ventricular mass indices were still significantly greater than in the control group (p < 0.01). Because of the incomplete regression of interventricular septal hypertrophy, the ratio between interventricular septum and posterior wall thickness was similar between both patient groups but it was significantly higher than in control subjects (p < 0.01). The ratio between wall thickness and chamber radius did not decrease significantly in group II patients, in whom it remained above the control values. CONCLUSION: Having a bileaflet aortic prosthesis of one size larger did not seem to significantly influence the pattern and the extent of regression of left ventricular hypertrophy after an intermediate period of follow-up.

Regression of left ventricular hypertrophy after aortic valve replacement for aortic stenosis with different valve substitutes.

OBJECTIVE: Stentless biologic aortic valves are less obstructive than stented biologic or mechanical valves. Their superior hemodynamic performances are expected to reflect in better regression of left ventricular hypertrophy. We compared the regression of left ventricular hypertrophy in 3 groups of patients undergoing aortic valve replacement for severe aortic stenosis. Group I (10 patients) received stentless biologic aortic valves, group II (10 patients) received stented biologic aortic valves, and group III (10 patients) received bileaflet mechanical aortic valves. METHODS: Echocardiographic evaluations were performed before the operation and after 1 year, and the results were compared with those of a control group. Left ventricular diameters and function, left ventricular wall thickness, and left ventricular mass were assessed by echocardiography. RESULTS: Group I patients had a significantly lower maximum and mean transprosthetic gradient than the other valve groups (P = .001). One year after operation there was a significant reduction in left ventricular mass for all patient groups (P < .01), but mass did not reach normal values (P = .05). Although the rate of regression in the interventricular septum and posterior wall thickness differed slightly among groups, their values at follow-up were comparable and still higher than control values (P = .002). The ratio between interventricular septum and posterior wall and the ratio between wall thickness and chamber radius did not change significantly at follow-up. CONCLUSIONS: Because the number of patients was relatively small, we could not use left ventricular mass regression after I year to distinguish among patients undergoing aortic valve replacement for aortic stenosis by means of valve prostheses with different hemodynamic performances.

Bentall procedure with a stentless valve and a new aortic root prosthesis.

We describe a technique to replace the aortic root by means of a stentless valve and a new aortic root Dacron graft (Gelweave Valsalva, Sulzer Vascutek, Renfrewshire, Scotland) that allows an anatomical reconstruction of the sinuses of Valsalva.

Opening and closing characteristics of the aortic valve after valve-sparing procedures using a new aortic root conduit.

BACKGROUND: The durability of aortic valve-sparing procedures is negatively affected by increased leaflet stress in the absence of normally shaped sinuses of Valsalva. We compared valve motion after remodeling procedures using a standard conduit and a specifically designed aortic root conduit. METHODS: Echocardiographic studies of the aortic valve dynamics were performed in 14 patients after remodeling of the aortic root (7 standard conduits, group A; 7 new conduits, group B) and in 7 controls (group C). Opening and closing leaflet velocities and percent of slow closing leaflet displacement were measured. Root distensibility and the pressure strain of the elastic modulus were measured at all root levels. RESULTS: Root distensibility and the pressure strain of the elastic modulus were different in group A and B only at the sinuses (p < 0.001). Opening and closing leaflet velocities were not different among groups. Slow closing leaflet displacement was markedly more evident in group B patients (24.2%+/-1.9% versus 2.5%+/-1.9% in group A, p < 0.001) and similar to controls (22.1%+/-7.9%). CONCLUSIONS: The new conduit guarantees dynamic features of the aortic valve leaflets superior to those obtained with standard conduits and more similar to normal subjects.

Residual aortic valve regurgitation after aortic root remodeling without a direct annuloplasty.

BACKGROUND: Aortic insufficiency secondary to degenerative aneurysms of the ascending aorta can be surgically treated with replacement of the valve or with remodeling of the aortic root. METHODS: In 15 patients who underwent aortic root remodeling from January 1994 to December 1996, we evaluated the postoperative aortic regurgitation and correlated it with several anatomic and functional variables. Operative success was defined as a residual aortic regurgitation less than or equal to 1 on a scale of 0 to 4. RESULTS: Root dimensions and aortic incompetence decreased significantly after the operation (p < 0.0001). The difference between preoperative and postoperative root diameters (p = 0.0006) and the presence of Marfan's syndrome (p < 0.0001) were independently predictive of persisting significant aortic insufficiency. Operative success was obtained in patients with a difference between preoperative and postoperative root diameters smaller than 30 mm. CONCLUSIONS: Aortic root remodeling is effective in reducing aortic regurgitation. Severe aortic root dilatation may result in excessive geometric alteration, leading to suboptimal results. The choice of a larger graft contributes to avoiding excessive geometric constraint of a profoundly diseased aortic root. Indication to undergo root remodeling should be evaluated cautiously in patients with Marfan's syndrome.