A. Corradi

Infection, immunity and the neuroendocrine response.

Abstract The Central Nervous (CNS) and Immune Systems (IS) are the two major adaptive systems which respond rapidly to numerous challenges that are able to compromise health. The defensive response strictly linking innate to acquired immunity, works continuously to limit pathogen invasion and damage. The efficiency of the innate response is crucial for survival and for an optimum priming of acquired immunity. During infection, the immune response is modulated by an integrated neuro-immune network which potentiates innate immunity, controls potential harmful effects and also addresses metabolic and nutritional modifications supporting immune function. In the last decade much knowledge has been gained on the molecular signals that orchestrate this integrated adaptive response, with focus on the systemic mediators which have a crucial role in driving and controlling an efficient protective response. These mediators are also able to signal alterations and control pathway dysfunctions which may be involved in the persistence and/or overexpression of inflammation that may lead to tissue damage and to a negative metabolic impact, causing retarded growth. This review aims to describe some important signalling pathways which drive bidirectional communication between the Immune and Nervous Systems during infection. Particular emphasis is placed on pro-inflammatory cytokines, immunomodulator hormones such as Glucocorticoids (GCs), Growth hormone (GH), Insulin-like Growth Factor-1 (IGF-1), and Leptin, as well as nutritional factors such as Zinc (Zn). Finally, the review includes up-to-date information on this neuroimmune cross-talk in domestic animals. Data in domestic animal species are still limited, but there are several exciting areas of research, like the potential interaction pathways between mediators (i.e. cytokine-HPA regulation, IL-6-GCS-Zn, cytokines-GH/IGF-1, IL-6-GH-Leptin and thymus activity) that are or could be promising topics of future research in veterinary medicine.

Targeting MEK/MAPK signal transduction module potentiates ATO-induced apoptosis in multiple myeloma cells through multiple signaling pathways

We demonstrate that blockade of the MEK/ERK signaling module, using the small-molecule inhibitors PD184352 or PD325901 (PD), strikingly enhances arsenic trioxide (ATO)-induced cytotoxicity in human myeloma cell lines (HMCLs) and in tumor cells from patients with multiple myeloma (MM) through a caspase-dependent mechanism. In HMCLs retaining a functional p53, PD treatment greatly enhances the ATO-induced p53 accumulation and p73, a p53 paralog, cooperates with p53 in caspase activation and apoptosis induction. In HMCLs carrying a nonfunctional p53, cotreatment with PD strikingly elevates the (DR4 + DR5)/(DcR1 + DcR2) tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors ratio and caspase-8 activation of ATO-treated cells. In MM cells, irrespective of p53 status, the combined PD/ATO treatment increases the level of the proapoptotic protein Bim (PD-mediated) and decreases antiapoptotic protein Mcl-1 (ATO-mediated). Moreover, Bim physically interacts with both DR4 and DR5 TRAIL receptors in PD/ATO-treated cells, and loss of Bim interferes with the activation of both extrinsic and intrinsic apoptotic pathways in response to PD/ATO. Finally, PD/ATO treatment induces tumor regression, prolongs survival, and is well tolerated in vivo in a human plasmacytoma xenograft model. These preclinical studies provide the framework for testing PD325901 and ATO combination therapy in clinical trials aimed to improve patient outcome in MM.

Zinc, thymic endocrine activity and mitogen responsiveness (PHA) in piglets exposed to maternal aflatoxicosis B1 and G1

Growth retardation, thymic involution and impaired peripheral immune efficiency are constant events in piglets exposed to maternal aflatoxicosis. Zinc may play a key role because of its requirement for good immune responses, including thymic endocrine activity. Zinc is required to activate a thymic hormone, i.e. thymulin (ZnFTS), which is responsible for cell-mediated immunity. Zinc deficiency and decreased thymic endocrine activity are present in piglets fed from sows exposed to aflatoxins (AF) B1 and G1 as compared with healthy control piglets. In particular, active ZnFTS is decreased while concentrations of inactive thymulin (FTS) are high. The in vitro addition of zinc up to the plasma samples induces a reduction of inactive thymulin. The lymphocytes mitogen responsiveness (PHA) is decreased and a thymic cortical lymphocyte depletion is also present. These data suggest that the thymic defect, followed by impaired peripheral immune efficiency, may largely depend by the low peripheral zinc bioavailability to saturate all thymulin molecules produced.

Isolation and characterization of lymphatic microvascular endothelial cells from human tonsils

Human lymphatic endothelial cells (LECs) have isolated prevalently from human derma and tumors. As specialized lymphatic organs within the oropharynx, palatine tonsils are easily obtained and rich in lymphatic venules. Using a two‐step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1 (UEA‐1)‐coated beads, followed by purification with monoclonal antibody D2–40, we successfully purified LECs from human palatine tonsils. The LECs were expanded on flasks coated with collagen type 1 and fibronectin for up to 8–10 passages and then analyzed for phenotypic and functional properties. Cultured cells retained the phenotypic pattern of the lymphatic endothelium of palatine tonsils and expressed functional VEGFR‐3 molecules. In fact, stimulation with VEGFR‐3 ligand, the vascular endothelium grow factor C, induced a marked increase in cell proliferation. Similarly to blood endothelial cells (BECs), LECs were able to form tube‐like structure when seeded in Cultrex basement membrane extract. Comparative studies performed on LECs derived from palatine tonsils and iliac lymphatic vessels (ILVs), obtained with the same procedures, showed substantial discrepancies in the expression of various lymphatic markers. This points to the existence of micro‐ and macrovessel‐derived LECs with different phenotypes, possibly involving different biological activities and functions. Palatine tonsil‐ and ILV‐derived LECs may, therefore, represent new models for investigating function and biochemical properties of these lymphatic endothelia. J. Cell. Physiol. 207: 107–113, 2006.

Experimental Tracheal Transplantation Using a Cryopreserved Aortic Allograft

Background: The tracheal reconstruction after wide resections remains a critical surgical problem. Our aim was to replace trachea with a tissue easy to vascularize, which allows a simple reconstruction and does not require an immunosuppressive regimen. Materials and Methods: A segment of cryopreserved aorta was used in order to verify its adequacy as tracheal substitute. In phase 1, the thoracic aorta of 10 rabbits was excised, obtaining 20 segments that were cryopreserved. Ten segments were implanted in the omentum of 10 rabbits that were sacrificed on postoperative days 7, 14 and 21, and the grafts were examined histologically. In phase 2, a segment of cryopreserved aorta arranged with a silicone prosthesis was transplanted in 10 rabbits and wrapped with omentum. The animals were sacrificed on postoperative days 7, 14 and 21. Results: In phase 1, the neovascularization of the grafts was present after 7 days, and after 14 days the fibroblasts invaded the lumen of the aorta. In phase 2, 8 rabbits survived and the histologic examination after 7, 14 and 21 days showed neovascularization, the absence of rejection and the proliferation of fibroblasts inside the lumen of the aorta; this growth has been restrained by an endoluminal prosthesis. Conclusions: Our study demonstrated that replacing the trachea with cryopreserved aorta is technically feasible and does not evoke immunologic reactions. It requires, however, a silicone tube inside the allograft to limit the colonization of fibroblasts.

Metallothionein (I+II) confers, via c-myc, immune plasticity in oldest mice: model of partial hepatectomy/liver regeneration

Abstract Because of its similarity to ageing in impaired immune efficiency 48 h after surgical procedures on young partially hepatectomised mice, partial hepatectomy/liver regeneration (pHx) provides a good model for the study of inflammation in ageing. In old age, high metallothionein (I+II) (MT) sequesters a substantial number of intracellular zinc ions consequently leading to low zinc ion bioavailability for an adequate immune response. Corticosterone and IL-6 affect MTmRNA induction in inflammation and after pHx against oxidative damage. The aim of this study was to investigate the role played by MT in conferring immune plasticity in ageing and in very old age using the pHx model. 48 h after their partial hepatectomy, the crude zinc balance was negative in young, old and very old mice coupled with increased MT, corticosterone, sIL-6R and IL-6. Concomitantly, Natural Killer (NK) cell activity and IL-2 production decreased. Complete restoration of the nutritional–endocrine–immune parameters occurred 15 days from the surgical procedures in young and very old mice, but not in old or transgenic mice overexpressing MT. A significant positive or inverse correlation among nutritional–endocrine–immune parameters exists in young and very old mice, but not in old mice during liver regeneration. Since MT also affects c-myc, the gene expression of c-myc declines from 48 h to days 7 and 15 after pHx in young and very old mice, but remains constantly high in old pHx mice for the same days. This circumstance leads to the appearance of tumours in the long run in old pHx mice and survival times that are shorter than old sham controls. Because complete remodelling also occurs in IL-6 and in sIL-6R in very old mice during liver regeneration, the pre-existing inflammation is not detrimental in very old age. As such, very old mice are still responsive to large inflammation, such as pHx, thanks to correct MT homeostasis. Correct MT homeostasis, via c-myc, is therefore pivotal in both suitable liver regeneration and in conferring immune plasticity with subsequent successful ageing. High MT plays an extremely harmful role in ageing: on one hand it lowers zinc ion bioavailability levels required for immune efficiency and on the other hand it increases c-myc expression. The combination of immune depression and enhanced c-myc, via high MT, may trigger the appearance of age-related degenerative diseases.

Attachment, proliferation and osteogenic response of osteoblast-like cells cultured on titanium treated by a novel multiphase anodic spark deposition process.

A new bioactive titanium surface treatment, labeled Ti-ASD, was developed using the electrochemical anodic spark deposition (ASD) technique and results in a thickened titanium oxide layer with higher levels of calcium and phosphorus typical of newly deposited mineral phase. This study was aimed at extending the knowledge on Ti-ASD treatment, by means of evaluation of the attachment, morphology, proliferation, metabolic activity, differentiation, and mineralization of osteoblast-like cells (SaOS-2) after growth on Ti-ASD treated titanium compared with nontreated titanium (Ti) and with chemically etched titanium (Ti-ETC). This novel type of titanium coating supported cell attachment, cell proliferation, and mineralization, revealing no cytotoxicity effects. The expression of differentiation markers on Ti-ASD treated titanium shows that genes related to the proliferation phase (Collagen type I, Coll I; Cbfa-1) were early expressed, whereas genes related to the mineralization phase (alkaline phosphatase, osteopontin, bone sialo protein) increased in a time-related way. Mineralization occurred on all analyzed surfaces, but on Ti-ASD the number of bone-like nodules and the amount of mineralized area was higher. In conclusion, Ti-ASD resulted to be a good surface for osteoblast attachment and proliferation, also promoting the maintenance of cell differentiation and matrix mineralization, a fundamental requirement for sustain the osseointegration and the clinical success of dental implants.

Innate pro-inflammatory and adaptive immune cytokines in PBMC of vaccinated and unvaccinated pigs naturally exposed to porcine circovirus type 2 (PCV2) infection vary with the occurrence of the disease and the viral burden.

Pro-inflammatory (IL-8, TNF-α, IL-1β) and immune (IFN-γ, IL-10) cytokines were evaluated in PCV2-vaccinated and unvaccinated pigs exposed to natural PCV2 infection retrospectively selected according to the time of the onset of viremia and the viral burden, and the presence of PMWS clinical signs. In a farrow-to-finish herd with a history of PMWS in animals aged older than 15 weeks, at weaning (21 ± 3 days of age), vaccinated pigs were intramuscularly inoculated with one dose of Porcilis(®) PCV vaccine+adjuvant whereas the adjuvant alone was administered to the control animals. Thirty animals bled at 16 weeks of age (before the occurrence of the natural infection and the onset of the disease) and then at 19, 20, 22 and 26 weeks of age, were categorized as: (a) vaccinated non-infected and non-PMWS-affected (PCV2-vac), (b) unvaccinated spontaneously infected/non-PMWS-affected (Ctrl) and (c) unvaccinated spontaneously infected/PMWS-affected (Ctrl-PMWS+) pigs. A major evidence of this study is that PMWS-affected animals were not able to mount an efficient innate pro-inflammatory response to cope with PCV2 infection as demonstrated by the low levels of pro-inflammatory cytokines, namely IL-8, TNF-α and IL-1β, and IFN-γ. Conversely, significantly increased gene expression levels of IL-8, TNF-α and IL-1β were detected especially in the PCV2-vac group at the early phase of the infection. Moreover, in PMWS diseased animals, a significant increase of IL-10 occurred at the early phase of infection, while, vaccinated pigs, in addition to the low viremia burden and its frequency and the absence of PMWS disease, showed a more stable IFN-γ response.

Prion protein genotypes of Italian sheep breeds with lysine-171 and phenylalanine-141 detection

Amino acid polymorphisms of the prion protein gene influence sheep susceptibility to classical and atypical scrapie. Substitutions at codons 136, 154 and 171 play an important role in classical scrapie. Codon 141 leucine to phenylalanine mutation (AFRQ) has been recognized as an increased risk factor for atypical scrapie. In addition a rare allele with lysine at codon 171 (ARK) has been detected in Mediterranean sheep breeds. The presence of ARK poses two problems: the determination of its frequency and its possible interference with genotyping output of routine methods lacking specific detection capacity for ARK. The aim of our work was the development of a routine genotyping method with the capacity to identify ARK and AFRQ in addition to the normally detected alleles and to determine the frequencies of all these alleles in 5 main Italian breeds: Sarda (n=2494), Bergamasca (n=2686), Appenninica (n=297), Comisana (n=361) and Massese (n=402). A multiplex primer extension assay targeting the six single nucleotide polymorphisms of interest was developed. Allele frequencies revealed a very low level of ARR in Bergamasca (6.91%) as opposed to the other breeds, very diverse levels of AFRQ ranging from absence in Comisana to 10.70% in Massese and a restricted presence of ARK. This allele has only been detected in Bargamasca with a significant 3.67% and marginally in Appenninica (0.34%). These results underline the need for adequate routine methods for genotyping of breeds with alleles that can interfere with typing of important codons such as the case of ARK for codon 171.

Adult Stem Cells: Perspectives for Therapeutic Applications

Ferrari, M., Corradi, A., Lazzaretti, M., De’Cillà, M., Losi, C.G., Villa, R. and Lanfranchi, A., 2007. Adult stem cells: Perspectives for therapeutic applications. Veterinary Research Communications, 31(Suppl. 1), 1–8AbstractThe use of adult stem cells in tissue regeneration appears to be a powerful research tool, due to the intrinsic characteristics of these cells, i.e., self-renewal and unlimited capacity for proliferation. In particular, mesenchymal stem cells (MSCs) obtained from bone marrow or peripheral blood can be easily isolated, cultivated, propagated and can be differentiated into several specialized cell types thanks to their plasticity. Among these cells, MSCs can evolve into cardiac cell lineages. Since heart damage leads to the irreversible loss of cardiac function, cell transplantation could be a potential therapy for heart injury. Our laboratory has focused on the purification and expansion of rat and sheep MSCs, their differentiation into cardiomyocytes and their characterisation. Numerous results indicate that MSCs could be promising for therapy, however we need to better understand the biology of stem cells to improve methods for delivery and/or pharmacological activation. These techniques can indeed track engrafted cells and systems to guarantee their safe use.