Investigations of some parameters of natural immunity in meat turkeys reared outdoors

The progression of some parameters of natural immunity in meat turkey reared outdoors was investigated. The levels of the haemolytic complement were lower than those obtained in commercial turkeys and in turkeys reared in a controlled environment during one of our previous investigations. The weather conditions could have influenced the trend of the haemolytic complement in turkeys selected for high meat production and kept outside, underlining the possible importance of the rearing system

Evaluation of Haematological and Immunological Parameters of the ASFV Lv17/WB/Rie1 Strain and Its Derived Mutant Lv17/WB/Rie1/d110-11L against ASFV Challenge Infection in Domestic Pigs

African swine fever virus (ASFV) is the etiological agent of a haemorrhagic disease that threatens the global pig industry. There is an urgency to develop a safe and efficient vaccine, but the knowledge of the immune–pathogenetic mechanisms behind ASFV infection is still very limited. In this paper, we evaluated the haematological and immunological parameters of domestic pigs vaccinated with the ASFV Lv17/WB/Rie1 strain or its derived mutant Lv17/WB/Rie1/d110-11L and then challenged with virulent Armenia/07 ASFV. Circulating levels of C-reactive protein (CRP), 13 key cytokines and 11 haematological parameters were evaluated throughout the study. Lv17/WB/Rie1 triggered an inflammatory response, with increased levels of CRP and pro-inflammatory cytokines, and induced lymphopenia, thrombocytopenia and a decline in red blood cell (RBC) parameters, although this was transitory. Lv17/WB/Rie1/d110-11L triggered only transitory thrombocytopenia and a mild inflammatory reaction, with no increase in serum levels of pro-inflammatory cytokines, but it raised IL-1Ra levels. Both strains counteracted several adverse reactions elicited by virulent challenge, like thrombocytopenia, a decline in RBC parameters, and inflammation. Within this paper, we provided a deep portrayal of the impact of diverse ASFV strains on the domestic pig’s immune system. A better understanding of these immune–pathological mechanisms would help to design suitable vaccines against this disease

PTEROATE-PEPTIDE BIOCONJUGATE TARGETING THE FOLATE RECEPTOR IN HUMAN OVARIAN CANCER CELL LINES: TRANSPORT AND MECHANISM OF ACTION.

Objectives The over-expression of thymidylate synthase (TS) and of the other folate cycle enzymes, is one of the mechanisms of resistance to cisplatin (cDDP) encountered in most of resistant human ovarian cancer cell lines, accounting for the more efficient DNA repair and synthesis. Oligopeptides were designed to inhibit TS activity by interfering with its dimerization. Among these, the LR octapeptide showed cell growth inhibitory activity against two cisplatin-sensitive human ovarian cancer cell lines. To improve the intracellular delivery of LR, we designed a bioconjugate with folic acid (FA-LR), which enters cell by exploiting the folate receptor alpha (FRα)-mediated endocytosis. Methods -Cell lines. The human ovarian cancer cell lines OAW28, COV504, IGROV-1, TOV112D, 2008, C13*, A2780 and A2780/CP. -Real Time PCR of FRα mRNA . -Flow cytometric analysis of FRα cell surface expression -Folic acid surface binding studies. -Uptake studies Results Real Time PCR, western blot analysis and folic acid surface binding assay indicate that IGROV-1 and OAW28 cells show high expression levels of FRα, while TOV112D, 2008 and 2008/C13* almost don't express FRα on their cell surface. The folate bioconjugate FA-LR blocked competitively the binding of [3H]Folic acid to FR and consequently its cellular uptake. FA-LR is detected in the cell and its stability evaluated. Conclusions The chemical modification of the folate with the LR drug motif only minimally altered the intrinsic affinity the biocongiugate for FR and suggest that the pteroate-peptide conjugate exploits FR as a substrate for its internalization. Cytotoxicity of the bioconjugate will be presented

PTEROATE-PEPTIDE BIOCONJUGATE TARGETING THE FOLATE RECEPTOR IN HUMAN OVARIAN CANCER CELL LINES: TRANSPORT AND MECHANISM OF ACTION.

Objectives The over-expression of thymidylate synthase (TS) and of the other folate cycle enzymes, is one of the mechanisms of resistance to cisplatin (cDDP) encountered in most of resistant human ovarian cancer cell lines, accounting for the more efficient DNA repair and synthesis. Oligopeptides were designed to inhibit TS activity by interfering with its dimerization. Among these, the LR octapeptide showed cell growth inhibitory activity against two cisplatin-sensitive human ovarian cancer cell lines. To improve the intracellular delivery of LR, we designed a bioconjugate with folic acid (FA-LR), which enters cell by exploiting the folate receptor alpha (FRα)-mediated endocytosis. Methods -Cell lines. The human ovarian cancer cell lines OAW28, COV504, IGROV-1, TOV112D, 2008, C13*, A2780 and A2780/CP. -Real Time PCR of FRα mRNA . -Flow cytometric analysis of FRα cell surface expression -Folic acid surface binding studies. -Uptake studies Results Real Time PCR, western blot analysis and folic acid surface binding assay indicate that IGROV-1 and OAW28 cells show high expression levels of FRα, while TOV112D, 2008 and 2008/C13* almost don't express FRα on their cell surface. The folate bioconjugate FA-LR blocked competitively the binding of [3H]Folic acid to FR and consequently its cellular uptake. FA-LR is detected in the cell and its stability evaluated. Conclusions The chemical modification of the folate with the LR drug motif only minimally altered the intrinsic affinity the biocongiugate for FR and suggest that the pteroate-peptide conjugate exploits FR as a substrate for its internalization. Cytotoxicity of the bioconjugate will be presented

Invited lecture to 18th World Congress on Advances in Oncology and 16th International Symposium on Molecular Medicine 10-12 October, 2013, Creta Maris, Hersonissos, Crete, Greece

Proteomic approach to the identification of early phase biomarker for anticancer peptides targeting thefolate pathway. F.Genovesea, A.Gualandia,b L.Taddiaa, G.Ponterinia, G.Marvertia, S.Pirondia, R.Guerrinic, M.Pelàa,c G.Pavesia, C.Trapellac, M.P.Costia aUniversity of Modena and Reggio Emilia, via Campi 183, 41125 Modena, Italy, bCRBA, S. Orsola University Hospital, Bologna, Italy, cDepartment of Pharmaceutical Science, University of Ferrara, Italy Many efforts to improve survival of patients affected by Ovarian Cancer (OC) have focused on more effective systemic therapies and on the search for new therapeutic targets. One of the molecular targets for OC is human Thymidylate Synthase (hTS), a homodimeric enzyme essential for DNA biosynthesis. In order to investigate the effects of hTS-interface-mimicking peptides at a cellular level, we started a study in which the cellular behavior of the peptides was investigated in combination with the proteomic differential analysis of the cytoplasmatic proteins of treated vs. untreated OC cells. The same experiment was performed with pemetrexed (PTX), a well known antifolate, for control purposes. The bioinformatic analysis of the effects of our peptide drug candidate indicates that deregulations can be mainly assigned to modulation of translational initiation, termination of RNA Pol-II transcription, transport, and protein catabolic events. Although apparently folate pathway members are not directly altered at a protein level, as the selection of ions to be sequenced is stochastic and biased towards abundant peptides, the bioinformatic analysis of peptide-modulated proteins suggested cellular investigations on the proteins of the folate-associated genes showing the largest number of dependencies to the species of the core set, which is required for the phosphorylation of several deoxyribonucleosides and nucleoside analogues. Comparison with the PTX-modulated proteins shows that some proteins of the proteasome complex and ribonucleoproteins are involved in both cases. These differences suggest that the two compounds may show a different mechanism of action which is in agreement with the hypothesized pharmacological model. Detailed cellular proteins profile based on the inferred roles of the identified proteins will further clarify the biological effects. 1.A proteomic approach to investigate the mechanism of action of anticancer peptides. F. Genovese, A. Gualandi, L. Taddia, M. Caselli, G. Ponterini, S.Ferrari, G. Marverti, R. Guerrini, M. Pela, G. Pavesi, C. Trapella, M.P. Costi, Proceeding 32EPS, p.466, ISBN 978-960-466-121-3). This work is supported by AIRC-DROC IG10474. www.unimore.airc-droc.i

Racemic synthesis and solid phase peptide synthesis application of the chimeric valine/leucine derivative 2-amino-3,3,4-trimethyl-pentanoic acid

The synthesis of non natural amino acid 2-amino-3,3,4-trimethyl-pentanoic acid (Ipv) ready for solid phase peptide synthesis has been developed. Copper (I) chloride Michael addition, followed by a Curtius rearrangement are the key steps for the Ipv synthesis. The racemic valine/leucine chimeric amino acid was then successfully inserted in position 5 of neuropeptide S (NPS) and the diastereomeric mixture separated by reverse phase HPLC. The two diastereomeric NPS derivatives were tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPS receptor where they behaved as partial agonist and pure antagonist

Assessment of BoAHV-1 Seronegative Latent Carrier by the Administration of Two Infectious Bovine Rhinotracheitis Live Marker Vaccines in Calves

Seronegative latent carriers (SNLCs) are animals that carry the virus without detectable antibodies and pose a risk for disease transmission and diagnostic challenges, suggesting the importance of consideration of marker vaccines in managing them. Therefore, in this study, we evaluated two modified live infectious bovine rhinotracheitis (IBR) marker vaccines (single and double deletions) for their ability to generate SNLC calves. These vaccines were administered to four groups (n = 3 in each group) of three-month-old calves in the presence or absence of passive immunity. Three hundred days after the first vaccination and after confirming the IBR seronegativity of all animals, dexamethasone was administered intravenously for five consecutive days. Only animals immunized with the modified live IBR marker vaccine (single deletion) in the absence of passive immunity exhibited a more enduring immune response than those vaccinated in the presence of passive immunity. Moreover, the administration of a modified live IBR marker vaccine (double deletion) to calves with passive immunity generated SNLC. These findings underscore the potential of live IBR marker vaccine (double-deletions) to aid serological diagnostic tools and develop vaccination protocols in achieving the desired immune response, particularly in the context of latent carrier status, offering valuable insights into optimizing vaccination strategies for effective IBR control

Evaluation of Safety and Efficacy of an Inactivated Marker Vaccine against Bovine alphaherpesvirus 1 (BoHV-1) in Water Buffalo (Bubalus bubalis)

Recent studies have explored the seropositivity of Bovine alphaherpesvirus 1 (BoHV-1) in water buffaloes, suggesting the urgency for developing strategies to eradicate the virus involving both cattle and water buffaloes. However, in Europe, the glycoprotein E (gE) deleted marker vaccines against BoHV-1 are commercially available only for the cattle industry. This study, for the first time, evaluated the safety and efficacy of a commercial inactivated gE-deleted marker vaccine in water buffalo. Five animals devoid of BoHV-1-neutralizing antibodies were vaccinated via intramuscular route. Five additional animals served as an unvaccinated control group. Sixty days after the first immunization, all animals were experimentally infected with a virulent BoHV-1via intranasal route. A detectable BoHV-1-humoral immune response was observed in the vaccinated group on post-vaccination day 30, whereas the antibodies appeared on post-challenge day 10 in the control group. Moreover, the vaccinated animals neither show viral shedding nor clinical signs compared to the control upon challenge. However, post-challenge, the BoHV-1-specific humoral and cell-mediated immune responses were significantly more increased in vaccinated animals than the control animals. Overall, the present study provides evidence of both the safety and efficacy of an inactivated gE-deleted marker vaccine against BoHV-1 in water buffaloes