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Abstract Background - Rabbits provide an excellent model for many animal and human diseases, such as cardiovascular diseases, for the development of new vaccines in wound healing management and in the field of tissue engineering of tendon, cartilage, bone and skin. The study presented herein aims to investigate the biological properties of bone marrow rabbit MSCs cultured in different conditions, in order to provide a basis for their clinical applications in veterinary medicine. Findings - MSCs were isolated from 5 New Zealand rabbits. Fold increase, CFU number, doubling time, differentiation ability and immunophenotype were analyzed. With the plating density of 10 cells/cm2 the fold increase was significantly lower with DMEM-20%FCS and MSCs growth was significantly higher with αMEM-hEGF. The highest clonogenic ability was found at 100 cell/cm2 with MSCBM and at 10 cell/cm2 with M199. Both at 10 and 100 cells/cm2, in αMEM medium, the highest CFU increase was obtained by adding bFGF. Supplementing culture media with 10%FCS-10%HS determined a significant increase of CFU. Conclusion - Our data suggest that different progenitor cells with differential sensitivity to media, sera and growth factors exist and the choice of culture conditions has to be carefully considered for MSC management.

Cigarette smoke is a mixture of chemicals having direct and/or indirect toxic effects on different lung cells. We investigated the effect of cigarette smoke on human lung fibroblasts (HFL-1) oxidation and apoptosis. Cells were exposed to various concentrations (1, 5, and 10%) of cigarette smoke extract (CSE) for 3 h, and oxidative stress and apoptosis were assessed by fluorescence-activated cell sorting and confocal laser fluorescence microscopy. Both oxidative stress and apoptosis exhibited a dose-response relationship with CSE concentrations. Lung fibroblasts also showed marked DNA fragmentation at the Comet assay after exposure to 10% CSE. Coincubation of HLF-1 cells with N-acetylcysteine (1 mM) during CSE exposure significantly reduced oxidative stress, apoptosis, and DNA fragmentation, whereas preincubation (3 h) with the glutathione-depleting agent buthionine sulfoximine (125 [micro]M) produced a significant increase of oxidative stress. Cigarette smoke is a potent source of oxidative stress, DNA damage, and apoptosis for HFL-1 cells, and we speculate that this could contribute to the development of pulmonary emphysema in the lungs of smokers. Comet assay

Polymorphism amongst the human leucocyte antigen (HLA) class II genes could influence antigen presentation and the ability to control human immunodeficiency virus (HIV)-1 by modulating the virus specific CD4 immune response. To examine the effect of such polymorphisms on disease progression, we studied a cohort of 46 HIV-1 infected long-term non-progressors (LTNPs), 87 intermediate progressors (IPs) and 26 rapid progressors. Kaplan­Meier survival analysis of all patients in the cohort on time to a CD4 count less than 350 cells/μl, showed a trend for a slower rate of CD4 decline in patients with, compared to those without, the DRB1*15–DQB1*06 haplotype (hazard ratio (HR) 0·69, 95% CI 0·46–1·01, P = 0·06). A similar effect was not observed with the DRB1*13–DQB1*06 haplotype (HR 1·18, 95% CI 0·75–1·88, P = 0·46), but was observed when DQB1*06 alleles were considered irrespective of their DR association (HR 0·74, 95% CI 0·52–1·05, P = 0·06). Major HLA-DQ6 alleles encode aspartate (Asp) at position 57 on the DQβ chain, a phenotype associated with protection from other immune disorders. We therefore examined the frequency of all DQβ57 Asp+ alleles, but could not detect a significant effect on the rate of CD4 decline. To examine whether the genotype associated with slower CD4 decline was over-represented in patients with a slow rate of disease progression, we conducted a categorical analysis of a subset of patients with an extended follow-up of 14+years. We found a higher proportion of LTNPs at 14+ years possessed the DRB1*15–DQB1*06 haplotype compared to IPs at 14+ years (38·46 versus 18·18%), though this difference did not reach statistical significance. When DQB1*06 alleles irrespective of their DR association were considered, the protective effect was greater (76·9% LTNPs versus 18·18% IPs, P = 0·04). Our results highlight the potential protective effect of HLA DQB1*06 alleles on the course of HIV disease. [ABSTRACT FROM AUTHOR]

To identify best growth conditions for the in vitro differentiation of human peripheral blood mono-nuclear cells (PBMNCs) into endothelial progenitor cells (EPC5), PBMNCs of healthy volunteers were cultured on fibronectin as follows: M199 with VEGE, bEGE, and IGF-I; M 199 with bovine retina-derived growth supplement (RDGS); human umbilical vein endothelial cell (HUVEC) conditioned medium; Dil-stained PBMNCs with HUVECs (1:4 ratio) in M 199 with RDGS (cocultures system); PHA burst (10μL/106 cells) for 24 h and culture in M199 with RDGF. EPCs were identified by FACS using mAbs for endothelial markers. EPCs migration was determined using a modified Boyden chamber assay and VEGF as chemoattractant. Matrigel was used to assess in vitro angiogenesis capability. Spindle-shaped and attached cells sprouted with growth factors, differentiating in EPCs within 2 weeks and forming cobblestone-like monolayers within 3 weeks. With RDGS, numerous large cell clusters appeared within 1 week but the number of clusters decreased during culture. After stimulation with PHA, many cells clusters and some adherent EC-like cells were observed within 7 days; their number and size increased within 14 days. With HUVEC conditioned medium, spindle-shaped cells were observed after 10 days. FACS confirmed the endothelial phenotype. Cocultures induced the appearance of double-labeled EPCs expressing endothelial antigens starting from 7 days. EPCs were able to migrate in response to VEGF and to incorporate in capillary-like structures formed by HUVECs on Matrigel. PBMNCs are able to differentiate in EPCs when stimulated with appropriate culture conditions. The contact with mature endothelial cells (ECs) makes this process quicker. [ABSTRACT FROM AUTHOR]

Cumulative cardiovascular risk, and cardiovascular disease status alter number and function of circulating endothelial progenitor cells (EPCs). Furthermore, circulating EPCs levels predict cardiovascular events. Diabetes reduces circulating EPCs level. In this scenario, our purpose was to investigate whether pre-diabetes impacts on progenitor cells (CPCs) and EPCs levels. A group of 27 pre-diabetic subjects (8 subjects with impaired fasting glucose, IFG; 14 with impaired glucose tolerance, IGT; and 5 with both IFG and IGT) and a group 22 control individuals have been recruited. No subject was on pharmacological treatments. BMI, waist, OGTT, HbA1c, blood pressure, lipids and inflammatory parameters were measured. Metabolic syndrome (MS) was assessed by NCEP/ATPIII criteria. CPCs (CD34+ cells) and EPCs (CD34+KDR+ cells) were assessed by FACS analysis. Data are expressed as cells number for ml by ISHAGE protocol and as EPCs/CPCs ratio. No differences were observed in EPCs and in EPCs/CPCs in pre-diabetics compared with controls (EPCs: 532±424 vs 511±348/ml, p=0.86; EPCs/CPCs: 28.0±16.1 vs 22.9±17.2%, p--0.30). No correlations were observed between EPCs or EPCs/CPCs and clinical (age, BMI, waist, blood pressure) or laboratory parameters (fasting or 2-h glucose, HbA1c, glucose area trader OGTT, lipids, hs-CRP). In pre-diabetic subjects, EPCs (516±298 vs 542±494/ml, p=0.88) and EPCs/CPCs (27.6±16.4 vs 28.3±16.4%, p=0.93) were superimposable in those with (n. 11, 40%) and without MS. All control subjects were free of MS. A positive first-degree family history for diabetes was present in 10 controls (45%) and 19 pre-diabetic subjects (70%). EPCs (436±334 vs 651±-432/ml, p=0.067) and EPCs/CPCs (21.9±14.3 vs 31.1± 18.5%, p=0.069) were lower in subjects with a family history for diabetes. In conclusions, minor impairments of glucose regulation (pre-diabetes) do not affect circulating EPCs. In pre-diabetic subjectss, EPCs levels are not modified by the co-occurrence of the Metabolic Syndrome. [ABSTRACT FROM AUTHOR]

Background: It has recently been demonstrated that zinc oxide nanoparticles (ZnO NPs) induce death of cancerous cells whilst having no cytotoxic effect on normal cells. However, there are several issues which need to be resolved before translation of zinc oxide nanoparticles into medical use, including lack of suitable biocompatible dispersion protocols and a better understanding being needed of the mechanism of their selective cytotoxic action., Methods: Nanoparticle dose affecting cell viability was evaluated in a model of proliferating cells both experimentally and mathematically. The key issue of selective toxicity of ZnO NPs toward proliferating cells was addressed by experiments using a biological model of noncancerous cells, ie, mesenchymal stem cells before and after cell differentiation to the osteogenic lineage., Results: In this paper, we report a biocompatible protocol for preparation of stable aqueous solutions of monodispersed zinc oxide nanoparticles. We found that the threshold of intracellular ZnO NP concentration required to induce cell death in proliferating cells is 0.4 ± 0.02 mM. Finally, flow cytometry analysis revealed that the threshold dose of zinc oxide nanoparticles was lethal to proliferating pluripotent mesenchymal stem cells but exhibited negligible cytotoxic effects to osteogenically differentiated mesenchymal stem cells., Conclusion: Results confirm the ZnO NP selective cytotoxic action on rapidly proliferating cells, whether benign or malignant.

Background: . The fact that only a small percentage of cord blood units (CBU) stored are actually used for transplantation contributes to raising the already high costs of their processing and cryopreservation. The identification of predictors allowing the early identification of suitable CBU would allow a reduction of costs for the collection, storage and characterisation of CBU with insufficient volume or cell numbers. In our bank we have adopted a cut-off value for using CBU of 8 x 10(8) nucleated cells and a volume >or= 60 mL., Materials and Methods: In 365 banked CBU, we evaluated the correlation between neonatal/gestational parameters and laboratory data used to assess their quality., Results: Biparietal diameter (BPD) and abdominal circumference were significantly and positively correlated with CBU volume (r(2)=0.12, p=0.0011 and r(2)=0.092, p=0.0063, respectively). Receiver operating characteristic (ROC) analysis showed that both parameters can be used to identify CBU with insufficient volume (BPD: area under the curve 0.69, 95% CI=0.57-0.82, p=0.004; abdominal circumference: area under the curve 0.67, 95% CI=0.54-0.79, p<0.01). BPD and head circumference, but not abdominal circumference or femoral length, were positively correlated with white blood cell (WBC) count (r(2)=0.215, p=0.031, and r(2)=0.299, p=0.015, respectively). Abdominal circumference, but not BPD, head circumference or femoral length, was statistically significantly correlated with the number of CD34(+) cells in the CBU. Weight at birth and placental weight were positively correlated with WBC count, blood volume, CD34(+) cell count, total colony-forming units and burst-forming units., Conclusion: . Pre-birth assessment of BPD might allow the selection of donors who would yield CBU of sufficient volume and WBC count and avoid the costs of collecting, transferring, storing and analysing CBU with a high probability of resulting unsuitable for transplantation.

Introduction: The aim of this study was to conduct a Regional survey to determine the policies and ways of performing the direct antiglobulin test in pre-transfusion screening, the approach used in cases giving positive results with this test and the technical and operative modalities for choosing blood for transfusion in cases of autoimmune haemolytic anaemia., Materials and Methods: A questionnaire, containing ten multiple-choice questions, was sent to all the transfusion centres in the Region of Tuscany., Results: The data from all 40 regional centres were analysed. Direct antiglobulin tests and autocontrols were not regularly used in pre-transfusion screening. The direct antiglobulin test was predominantly reserved for suspected cases of autoimmune haemolytic anaemia. Sixty percent of the laboratories characterised the specificity of samples that were positive for IgG and complement by the direct antiglobulin test, 45% that were positive for IgM, 35% also for IgA, and 13% also for subclasses of IgG. Elution studies were reserved (in 18% of laboratories) for those cases in which it was expected that transfusion therapy would be used. In cases of autoimmune haemolytic anaemia, autologous/allogeneic adsorption was carried out in 27% of the structures (the use of proteolytic enzymes is predominant, followed by the "ZZAP" reagent--a mixture of dithiothreitol and an enzyme) and the dilution method in 20%; transfusion of red blood cells with a phenotype extensively compatible (c, C, D, E, e, K, Jka, Jkb, Fya, Fyb, S, s) with that of the recipient is practised in 17% of the centres, while transfusion of units of "least incompatible" red blood cells was reported by 95% of the centres, but in 88% this is preceded by at least one of the above mentioned immunohaematological investigations., Conclusions: The organisation of a network of Services of Immunohaematology and Transfusion Medicine can be exploited to overcome some technical and operative limitations of peripheral, dependent Transfusion Sections. The results of this study reveal which immunohaematology laboratory is endowed with the greatest potential and which could, therefore, become the regional reference centre. This investigation could lay the basis for defining behavioural algorithms and recommendations on the issues considered.

GH has antiapoptotic effects on several cells. However, the antiapoptotic mechanisms of GH on colonic mucosa cells are not completely understood. Peroxisome proliferator activated receptor-gamma (PPARgamma) activation enhances apoptosis, and a link between GH and PPARgamma in the colonic epithelium of acromegalic patients has been suggested. We investigated the effects of GH and of PPARgamma ligands on apoptosis in colonic cancer cell lines. Colonic cells showed specific binding sites for GH, and after exposure to 0.05-50 nm GH, their apoptosis reduced by 45%. The antiapoptotic effect was due to either GH directly or GH-dependent local production of IGF-1. A 55-85% reduction of PPARgamma expression was observed in GH-treated cells, compared with controls (P < 0.05). However, treatment of the cells with 1-50 microm ciglitazone (cig), induced apoptosis and reverted the antiapoptotic effects of GH by increasing the programmed cell death up to 3.5-fold at 30 min and up to 1.7-fold at 24 h. Expression of Bcl-2 and TNF-related apoptosis-induced ligand was not affected by either GH or cig treatment, whereas GH reduced the expression of Bax, which was increased by cig treatment. In addition, GH increased the expression of signal transducer and activator of transcription 5b, which might be involved in the down-regulation of PPARgamma expression. In conclusion, GH may exert a direct antiapoptotic effect on colonic cells, through an increased expression of signal transducer and activator of transcription 5b and a reduction of Bax and PPARgamma. The reduced GH-dependent apoptosis can be overcome by PPARgamma ligands, which might be useful chemopreventive agents in acromegalic patients, who have an increased colonic polyps prevalence.