After more than 10 years of absence, sylvatic rabies re-appeared in Italy in 2008. To prevent disease spread, three oral rabies vaccination (ORV) campaigns targeting red foxes were performed through manual distribution of vaccine baits between January and September 2009. As these campaigns proved unsuccessful, at the end of December 2009, baits started being distributed using helicopters, allowing uniform coverage of larger areas in a shorter time period. From winter 2009 to autumn 2016, a total of 15 ORV campaigns (four emergency, four regular and seven preventive ORV) were implemented through aerial distribution of baits. In this study, we assessed the costs of the aerial ORV campaigns, which were aimed at eradicating the disease and reobtaining the rabies-free status. Cumulative costs per km2 were estimated at €59.45 during emergency campaigns and ranged between €51.94 and €65.67 in the regular vaccinations. The main portion of costs for ORV programmes were related to baits supply and distribution: €49.24 (82.83%) in emergency campaigns and from €40.33 to € 43.35 in regular ORVs (71.97% and 66.02%, respectively). At the end of each ORV campaign, the efficacy of vaccination activities was estimated by assessing the proportion of foxes testing positive for tetracycline biomarker in jawbone, indicating bait intake. Results revealed that the proportion of foxes that ingested baits varied between 70.97% and 95.51%. Statistical analysis indicated that reducing the density of dropped baits could potentially lead to a cost-saving of 22.81%, still maintaining a satisfactory level of bait intake by the fox population.

In 1999, Italy experienced a devastating epidemic of high-pathogenicity avian influenza (HPAI) caused by an H7N1 virus subtype. After this epidemic, a ministerial decree was passed to implement control measures for low-pathogenicity avian influenza (LPAI) due to H5 and H7 subtypes. We investigated whether these control measures have decreased the public expenditure associated with epidemics of LPAI and HPAI by comparing the direct and consequential losses of the 1999 epidemic to the losses associated with successive epidemics. The estimated total economic burden of the epidemics was about euro650 million (euro217 million in direct losses and euro433 million in consequential losses). The 1999 epidemic accounted for most of these losses (euro507 million: euro112 million in direct losses and euro395 million in consequential losses), whereas the total economic burden for the 5 successive LPAI was euro143 million (euro105 million in direct losses and euro38 million in consequential losses). These results demonstrate that the implementation of a coordinated set of disease-control measures, which included both emergency and prophylactic vaccination, was able to reduce the overall costs associated with avian influenza epidemics. The results also show that the application of adequate LPAI control measures may limit the risk of emergence of an HPAI virus in an area with a high poultry density, allowing the complete disruption of the poultry market and its huge associated costs to be avoided.

Transplanting hematopoietic and peripheral blood-derived stem/progenitor cells can have beneficial effects in slowing the effects of heart failure. We investigated whether human bone marrow CD133 + -derived cells (BM-CD133 + cells) might be used for cell therapy of heart injury in combination with tissue engineering. We examined these cells for: 1) their in vitro capacity to be converted into cardiomyocytes (CMs), and 2) their potential for in vivo differentiation when delivered to a tissue-engineered type I collagen patch placed on injured hearts (group II). To ensure a microvascular network ready for use by the transplanted cells, cardiac injury and patching were scheduled 2 weeks before cell injection. The cardiovascular potential of the BM-CD133 + cells was compared with that of a direct injection (group I) of the same cells in heart tissue damaged according to the same schedule as for group II. While a small fraction (2 ± 0.5%) of BM-CD133 + cells cocultured with rat CMs switched in vitro to a CM-like cell phenotype, in vivo—and in both groups of nude rats transplanted with BM-CD133 + —there was no evidence of any CM differentiation (as detected by cardiac troponin I expression), but there were signs instead of new capillaries and small arterioles. While capillaries prevailed over arterioles in group II, the opposite occurred in group I. The transplanted cells further contributed to the formation of new microvessels induced by the patch (group II) but the number of vessels did not appear superior to the one developed after directly injecting the BM-CD133 + cells into the injured heart. Although chimeric human–rat microvessels were consistently found in the hearts of both groups I and II, they represented a minority (1.5–2.3%) compared with those of rat origin. Smooth muscle myosin isoform expression suggested that the arterioles achieved complete differentiation irrespective of the presence or absence of the collagen patch. These findings suggest that: 1) BM-CD133 + cells display a limited propensity for in vitro conversion to CMs; 2) the preliminarily vascularized bioscaffold did not confer a selective homing and differentiation advantage for the phenotypic conversion of BM-CD133 + cells into CMs; and 3) combined patching and cell transplantation is suitable for angiogenesis and arteriogenesis, but it does not produce better results, in terms of endothelial and smooth muscle cell differentiation, than the “traditional” method of cell injection into the myocardium.

A reduction in the number of endothelial progenitor cells (EPCs) is considered a plausible cause of increased cardiovascular risk in diabetes mellitus. The aim of this study was to test the hypothesis that weak bone marrow mobilisation is responsible for the decrease in circulating EPCs in diabetes.

An unexplained paradox puzzles diabetologists: diabetic patients must face both poor vessel growth in ischemic heart and limbs and increased angiogenesis in retinal complications (1,2). Endothelial progenitor cells (EPCs) are marrow-derived cells involved in adult neovascularization and endothelial homeostasis (3,4). It has been postulated that low EPCs in peripheral blood may have a role in cardiovascular disease, and we have demonstrated that EPCs are reduced in macrovascular diabetes complications (5,6). On the other hand, an excess of EPCs may be involved in pathologic neoangiogenesis of cancer and proliferative retinopathy (7,8). Therefore, diabetes complications may be associated with both decreased and increased EPCs. Recently, novel therapeutic approaches have been directed to enrich the EPC pool in ischemic diseases and to block EPC function in proliferative diseases (9,10). These approaches in diabetic subjects require cautious evaluation of the implications carried by the paradox and new studies to unravel its causes (11,12). This study was carried out to investigate the contemporaneous effects of retinal and peripheral vascular complications on circulating progenitor cells. Ethics committee approval was obtained, and after giving informed consent, 60 type 2 diabetic patients were prospectively included. Patients were characterized in terms of peripheral arterial disease (PAD) and diabetic retinopathy (DR) as the most representative complications at the two extremities of the diabetic paradox. PAD was diagnosed by minimal criteria (including history of claudication or rest pain, pulse examination, ankle-brachial indexes, and ultrasonography) and eventually confirmed by angiography. Diabetic retinopathy (severe nonproliferative or proliferative) was defined by a dilated and comprehensive eye examination and acquisition of high-quality stereoscopic photographs by …

Objective: Transplantation of stem cells in the acute ischemic myocardium (AMI) may play a rote in the recovery of cardiac function. Here, we investigated the ability of amniotic fluid-derived mesenchymal cells (AFC) for phenotypic conversion to vascular cells and cardiomyocytes (CM) when autotransplanted in a porcine model of AMI. Methods: Single AFC preparations were taken from 12 fetuses 3 days before normal delivery. AFC were expanded in vitro and stored separately until animals of the original litter weighed 22-25 kg. A new model of AMI, i.e. 45-min circumflex coronary occlusion followed by watt dissection, was used to assess AFC differentiation potential. CMFDA-labeled AFC were autogenically transplanted in the ischemic area I week after AMI induction. Thirty days Later, pigs were sacrificed and the phenotypic profile of transplanted AFC was assessed and compared to the corresponding pre-injection pattern. Results: AFC showed in vitro to be of mesenchymal type also expressing markers of 'embryonic stem' cells (SSEA4 and Oct-4), as well as endothelial (von Willebrand factor, VE-cadherin) and smooth muscle (SM alpha-actin, SM22) cells. Thirty days after transplantation, in the survived AFC (5 +/- 1%) 'embryonic stem' cell markers disappeared and mesenchymal cell markers were down regulated with the exception of smooth muscle and endothelial antigens. No evidence for expression of cardiac troponin I was found. Conclusions: In the conditions used in this study, AFC were able to transdifferentiate to cells of vascular cell lineages but not to CM. Thus, porcine AFC may require further ex vivo re-programming to be suitable for therapeutic use in AMI. (c) 2005 Elsevier B.V. ALL rights reserved.

Significant structural and functional changes in smooth muscle cells (SMCs) of microvessels (diameter 30 to 300 microm) occur in hypertension. However, in microvessels of hypertensive patients, the differentiation pattern of SMCs underlying such changes remains undefined. To analyze the differentiation pattern of SMCs (adult, postnatal, or fetal), 49 muscle biopsies (rectus abdominis) were analyzed: 16 from children (aged 11 months to 11 years), 15 from normotensive adults (aged 55 to 74 years), 18 from hypertensive adults (aged 55 to 74 years). Transverse cryosections of specimens were studied by immunocytochemistry using monoclonal antibodies SM-E7 and NM-F6, which recognize smooth muscle myosin heavy chain (MyHC) and A(pla1)-like nonmuscle MyHC, respectively. The total number of microvessels was assessed via SM-E7 staining. The number of NM-F6 positive (fetal-type SMC) or negative (adult-type SMC) microvessels was assessed. The number of microvessels per area unit was considerably lower (P<0.0005) in normotensive adults (0.22+/-0.17) than in children (0.98+/-0.61). Even more significant reduction was found in hypertensive adults compared with control adults (P=0.013) and children (P<0.0005). The qualitative immunocytochemistry analysis by NM-F6 revealed 2 differentiation patterns of the media layer of microvessels: positive or negative. In hypertensive subjects, the percentage of microvessels positive to NM-F6 was 49.8+/-35.6%, close to that found in children (50.6+/-12.6%), whereas in normotensive subjects it was significantly lower (24.4+/-21.1%). The following conclusions were drawn. (1) The medial layer of microvessels is heterogeneous in terms of SMC differentiation. (2) In hypertension, a prevalence of fetal-type SMCs takes place in microvessels, resembling that of children. Compared with children, a rarefaction of microvessels is present in normotensive adults that is even more remarkable in hypertensive adults.

Purpose: To investigate the role of smooth muscle cell (SMC) response and platelet activation in peripheral venous catheterization using a model of catheter injury associated with thrombocytopenic treatment. Methods: Silicon elastic catheters were inserted into New Zealand White rabbit external jugular veins from 24 hours to 60 days. Immunocytochemical procedures with antibodies to differentiation markers specific for SMCs, myofibroblasts, and endothelial cells were used to ascertain the phenotypic features of injured venous SMCs and the tissue sleeve formed around the catheter. Thrombocytopenia was induced in rabbits by busulfan treatment and the effect on catheter injury development examined after 15 days. The putative direct effect of this drug on the venous SMC proliferation, migration, and differentiation was assayed in vitro for 48 hours. Results: Catheter injury is characterized by the progressive formation of (1) a neointima, containing differentiating SMCs, which are derived from the media and adventitial layer, and (2) by the organizing thrombus formed around the catheter, which contains myofibroblasts. In busulfan-treated thrombocytopenic animals, there was no evidence for either neointimal development or thrombus formation. A direct role of this drug in the unresponsiveness of vascular wall can be excluded by the unchanged proliferation and migration pattern of cultured venous SMCs treated with busulfan compared to control cultures. Conclusions: In our model, accumulation of differentiated SMCs in the neointima and myofibroblast appearance in the thrombus are linked, although distinct, events regulated by platelet activation, which is able to furnish the appropriate microenvironment for vascular SMC recruitment from the media/adventitial layer.

BACKGROUND Fechtner syndrome (FTNS), also known as Alport-like syndrome, is a rare inherited condition characterized by progressive nephritis, macrothrombocytopenia, Döhle-like leukocyte inclusions, deafness, and cataract. Although it recently was shown that FTNS derives from mutation of MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA), its pathophysiological characteristics remain unknown. METHODS We studied a large FTNS family in which 10 components carried a missense mutation of MYH9 determining the D1424H substitution. RESULTS All affected subjects presented with macrothrombocytopenia and leukocyte Döhle-like bodies consisting of macroaggregates of NMMHC-IIA, but only two subjects had major renal problems characterized by proteinuria and renal failure. Electron microscopy showed focal and segmental effacement of podocytes and loss of the interpodocyte slit diaphragm. Immunohistochemistry showed apical localization of NMMHC-IIA in tubular epithelia and less podocyte staining in the two patients, whereas it was diffuse in normal epithelia. Three patients presented with stable microhematuria, and another five patients had no renal lesions, although they carried the same mutation of MYH9. Therefore, MYH9 mutation per se was responsible for platelet and leukocyte abnormalities, whereas additional predisposing conditions and/or environmental factors are necessary for nephropathy, cataract, and deafness. Looking at podocyte components conferring permselectivity properties to the kidney, we characterized the haplotype of podocin and found cosegregation of one specific allele in the two patients with nephrotic syndrome, suggesting a relationship between podocin features and proteinuria. CONCLUSION Our study indicates a major role for the NMMHC-IIA abnormality in the pathogenesis of leukocyte, platelet, and kidney defects in FTNS. The basic feature in all cases is aggregation and compartmentation of NMMHC-IIA. However, proteinuria and podocyte lesions are the hallmark of nephropathy in patients who develop renal failure, and podocin may have some function in this setting.

BackgroundHeart valve bioprostheses for cardiac valve replacement are fabricated by xeno- or allograft tissues. Decellularization techniques and tissue engineering technologies applied to these tissues might contribute to the reduction in risk of calcification and immune response. Surprisingly, there are few data on the cell phenotypes obtained after cellularizing these naturally-derived biomaterials in comparison to those expressed in the intact valve.MethodsAortic valve interstitial cells (VIC) were used to repopulate the corresponding valve leaflets after a novel decellularization procedure based on the use of ionic and nonionic detergents. VIC from leaflet microexplants at the third passage were utilized to repopulate the decellularized leaflets. Intact, decellularized and repopulated valve leaflets and cultured VIC were examined by immunocytochemical procedures with a panel of antibodies to smooth muscle and nonmuscle differentiation antigens. Intact and cellularized leaflets were also investigated with Western blotting and transmission electron microscopy, respectively.ResultsMyofibroblasts and smooth muscle cells (SMC) were mostly localized to the ventricularis of the leaflet whereas fibroblasts were dispersed unevenly. Cultured VIC were comprised of myofibroblasts and fibroblasts with no evidence of endothelial cells and SMC. Two weeks after VIC seeding into decellularized leaflets, grafted cells were found penetrating the bioscaffold. The immunophenotypic and ultrastructural properties of the grafted cells indicated that a VIC heterogeneous mesenchymal cell population was present: fibroblasts, myofibroblasts, SMC, and endothelial cells.ConclusionsVIC seeding on detergent-treated valve bioscaffolds has the cellular potential to reconstruct a viable aortic valve.

Summary. May–Hegglin anomaly (MHA), Sebastian syndrome (SBS) and Fechtner syndrome (FTNS) are autosomal‐dominant macrothrombocytopenias with Döhle‐like leucocyte inclusions. These diseases are due to mutations of the MHY9 gene, encoding the heavy chain of non‐muscle myosin IIA (NMMHC‐A). We investigated the NMMHC‐A localization in blood cells from eight MHA, SBS or FTNS patients with known MYH9 mutations. All the patients showed an altered localization of NMMHC‐A in granulocytes and platelets, suggesting that Döhle‐like bodies are due to the aggregation of NMMHC‐A in the cytoplasm. Therefore, immunocytochemistry for NMMHC‐A is a simple and sensitive method to detect pathological phenotypes of granulocytes and platelets in the diagnosis of MYH9‐related disorders.

The importance of mononuclear leukocyte (MO) adhesion to dysfunctional endothelium and migration to the subendothelial space in the early phases of atherogenesis is well established. Few studies have addressed the relevance of polymorphonuclear leukocytes (PMNs) in the context of evolving lesions, and nothing is known about PMN interaction with vascular smooth muscle cells (SMCs). In this study, we investigated leukocyte/SMC interactions in a model of rabbit carotid injury induced by placement of a silastic collar. This procedure leads to the development of intimal thickening characterized by SMC accumulation preceded by an abundant leukocyte infiltration. By transmission electron microscopy and immunocytochemistry, we demonstrated the occurrence of PMN infiltration starting at 6 h and ceasing within 72 h after collar placement. A previously unknown extensive interaction between medial myocytes and PMNs was detected, referring to emperipolesis, an active phenomenon of cells engulfing other cells in a process other than phagocytosis. PMNs, but not MOs, were internalized by SMCs, which showed ultrastructural features intermediate between the true contractile and the fully synthetic phenotype without exhibiting any sign of injury. Emperipolesis preceded any detectable cell proliferation in the vessel wall and disappeared within 72 h, following the kinetic of PMN infiltration in the vessel wall. In summary, our findings show the occurrence of an active and selective interaction between PMNs and SMCs via emperipolesis during the early phases of intimal thickening after perivascular collaring. However, the overall etiology of the phenomena described in the present study and their pathophysiological significance should be further investigated.

lBackground: We investigated the effect of nifedipine, AT-1 and ET-1 receptor blockade oil arterial smooth muscle cell phenotypes and collagen deposition in TGRen2 transgenic rat (TGR). Methods: Four-week-old TGR were blood pressure (BP)-matched and allocated to receive a placebo (n=8). the calcium antagonist nifedipine (n=6), the AT-1 specific receptor antagonist irbesartan (n=6), the ETA/ETB antagonist bosentan (n=6) or the ETA-selective antagonist BMS-182874 (n=5). Sprague-Dawley normotensive rats served Lis controls (n=6). After 4 weeks of treatment animals Were euthanized and the left ventricle (LV) and the structural changes in intracardiac arterioles and aorta were assessed histomorphometrically. Smooth muscle cell phenotypes and fibrillar collagen content of the aortic wall were evaluated by immunostaining, using differentiation markers-specific antibodies and Syrius red staining, respectively. The changes in ETA and ETB receptor density were also assessed with quantitative autoradiography. Results: Compared to placebo. only irbesartan lowered BP (P<0.001) and prevented LV and small resistance artery hypertrophy. The aorta of placebo-treated TGR showed all increase in foctal-type smooth muscle cell content and fibrillar collagen staining, compared to controls. These changes were blunted by irbesartan, which increased ETA receptors in the arterial wall, enhanced by BMS-182874 and unaffected by bosentan. Nifedipine also blunted both the VSMC and collagen changes despite having no effect oil BP and ETA receptors. Conclusions: In TGRen2, vascular hypertrophy entails both smooth muscle cell phenotypic modulation and collagen deposition. These alterations do not follow closely the BP challges and seem to imply the dihydropyridine-sensitive calcium channels. (C) 2002 Elsevier Science B.V. All rights reserved.

The adventitial layer surrounding the blood vessels has long been exclusively considered a supporting tissue the main function of which is to provide adequate nourishment to the muscle layers of tunica media. Although functionally interconnected, the adventitial and medial layers are structurally interfaced at the external elastic lamina level, clearly distinguishable at the maturational phase of vascular morphogenesis. Over the last few years the "passive" role that the adventitia seemed to play in experimental and spontaneous vascular pathologies involving proliferation, migration, differentiation, and apoptosis of vascular smooth muscle cells (VSMCs) has been questioned. It has been demonstrated that fibroblasts from the adventitia display an important partnership with the resident medial VSMCs in terms of phenotypic conversion, proliferation, apoptotic, and migratory properties the result of which is neointima formation and vascular remodeling. This article is an attempt at reviewing the major themes and more recent findings dealing with the phenotypic conversion process that leads adventitial "passive" (static) fibroblasts to become "activated" (mobile) myofibroblasts. This event shows some facets in common with vascular morphogenesis, ie, the process of recruitment, incorporation, and phenotypic conversion of cells surrounding the primitive endothelial tube in the definitive vessel wall. We hypothesize that during the response to vascular injuries in the adult, "activation" of adventitial fibroblasts is, at least in part, reminiscent of a developmental program that also invests, although with distinct spatiotemporal features, medial VSMCs.

57 Arterial hypertension is associated with vascular smooth muscle cells (VSMC) phenotypic differentiation, but the role of Ang II and ET-1 is still unclear. Thus, we investigated the changes of VSMC phenotypes in Ang II-dependent hypertension and the role of ET-1 and its A receptor (ET A ). Four-week old heterozygote male TGR rats (n=24) were body weight (BW)- and blood pressure (BP)-matched and randomly allocated to receive orally a placebo (Group P), the mixed ET A /ET B antagonist Bosentan (100 mg/Kg, Group B) the Ang II AT-1 specific receptor antagonist Irbesartan (50 mg/Kg BW, Group I) or the ET A selective antagonist BMS-182874 (52 mg/Kg BW, Group BMS). After 4-wk of treatments, during which BW and BP were measured weekly, animals were euthanized; the iliac artery and mesenteric arterioles were collected. In the latter the structural changes were assessed with a myograph. Immunohistochemistry with a panel of different antibodies specific for ET-1, ET A , smooth muscle (SM) myosin, SM actin, SM 22, myosin heavy chains Apla 22 and fibronectin EIIIA was carried out. The fetal, neonatal and adult aorta from normotensive Sprague-Dawley rats were studied as control. Compared to all other groups, Group I rats showed significantly (p<0.001) lower systolic BP (161±8 mmHg, vs 269±23 Group P; vs 254±21 Group BMS), LV weight (2.28±0.15 mg/g BW vs 3.71±0.26, 3.38±0.27 and 3.96±0.51), and normalized media thickness of the mesenteric arterioles (22.3±0.6 μm vs 25.3±0.5, 25.5±0.7 and 24.1±1.5). Hypertension, LVH and medial arterioles hypertrophy in group P TGR were paralleled by a shift of VSMC toward a fetal phenotype in the iliac artery, despite no change in the expression of both irET-1 and ET A . The VSMC phenotypic shift was prevented by both irbesartan and Bosentan, but not by the ET A -selective antagonist BM 182874. Thus, Ang II-dependent hypertension of TGR is associated with both vascular hypertrophy and a shift of VSMC toward a fetal phenotype, which occurs through AT-1- and ET B - but not ET A -mediated mechanisms.