In vivo testing of novel vaccine prototypes against Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is a Gram-negative bacterium that represents the main cause of porcine pleuropneumonia in pigs, causing significant economic losses to the livestock industry worldwide. A. pleuropneumoniae, as the majority of Gram-negative bacteria, excrete vesicles from its outer membrane (OM), accordingly defined as outer membrane vesicles (OMVs). Thanks to their antigenic similarity to the OM, OMVs have emerged as a promising tool in vaccinology. In this study we describe the in vivo testing of several vaccine prototypes for the prevention of infection by all known A. pleuropneumoniae serotypes. Previously identified vaccine candidates, the recombinant proteins ApfA and VacJ, administered individually or in various combinations with the OMVs, were employed as vaccination strategies. Our data show that the addition of the OMVs in the vaccine formulations significantly increased the specific IgG titer against both ApfA and VacJ in the immunized animals, confirming the previously postulated potential of the OMVs as adjuvant. Unfortunately, the antibody response raised did not translate into an effective protection against A. pleuropneumoniae infection, as none of the immunized groups following challenge showed a significantly lower degree of lesions than the controls. Interestingly, quite the opposite was true, as the animals with the highest IgG titers were also the ones bearing the most extensive lesions in their lungs. These results shed new light on A. pleuropneumoniae pathogenicity, suggesting that antibody-mediated cytotoxicity from the host immune response may play a central role in the development of the lesions typically associated with A. pleuropneumoniae infections

Quantitative differences in TGF-β family members measured in small antral follicle fluids from women with or without PCO

CONTEXT: Members of the Transforming-Growth-Factor-β (TGF-β) family have been implicated in aberrant follicle development in women with polycystic ovaries (PCO). OBJECTIVE: Are there quantitative differences in the concentrations of TGF-β family members in fluid from small antral follicles (hSAF) from women with or without PCO? DESIGN SETTING: & Follicle fluids (FF) were collected from 4-11 mm hSAF obtained from women undergoing ovarian tissue cryopreservation for fertility preservation. PATIENTS: FFs from 16 women with PCO (FF=93) and 33 women without PCO (FF=92). MAIN OUTCOME MEASURES: Intrafollicular concentrations of Growth-Differentiation-Factor-9 (GDF9), Anti-Müllerian-Hormone (AMH), inhibin-A and -B, total inhibin, activin-A, -B and -AB, follistatin, follistatin-like-3, estradiol, and testosterone. RESULTS: Activin-B concentrations are reported for the first time in hSAF and concentrations were 10 times higher than activin-A and -AB. Activin-B showed significant associations to other growth factors. Concentrations of inhibin-A and -B were significantly lower in FF from women with PCO, especially in hSAF below 8 mm in diameter. AMH concentrations did not differ between the groups in hSAF below 8 mm, however, AMH remained high in hSAF above 8 mm in PCO but decreased in non-PCO women. Estradiol was significantly lower in FF from women with PCO and showed significant associations with AMH. Concentrations of GDF9 are reported for the first time showing significantly higher concentrations in PCO FF of follicles above 6 mm. CONCLUSIONS: Altered concentrations of TGF-β family members in hSAF from women with PCO highlight altered growth factor signaling as a potential mechanism for follicle growth arrest

Intrafollicular concentrations of the oocyte-secreted factors GDF9 and BMP15 vary inversely in polycystic ovaries

Context The oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) play essential roles in follicle development and oocyte maturation, and aberrant regulation might contribute to the pathogenesis of polycystic ovary syndrome. Objective Are there measurable differences in concentrations of GDF9, BMP15, and the GDF9/BMP15 heterodimer in small antral follicle fluids from women with and without polycystic ovaries (PCO)? Design and Setting Follicle fluids (n = 356) were collected from 4- to 11-mm follicles in unstimulated ovaries of 87 women undergoing ovarian tissue cryopreservation for fertility preservation. Patients Twenty-seven women with PCO were identified and 60 women without PCO-like characteristics (non-PCO women) were matched according to age and follicle size. Main outcome measures Intrafollicular concentrations of GDF9, BMP15, GDF9/BMP15 heterodimer, anti-Mullerian hormone (AMH), inhibin-A and -B, total inhibin, activin-B and -AB, and follistatin were measured using enzyme-linked immunosorbent assays. Results The detectability of GDF9, BMP15, and the GDF9/BMP15 heterodimer were 100%, 94.4%, and 91.5%, respectively, and concentrations were significantly negatively correlated with increasing follicle size (P < 0.0001). GDF9 was significantly higher in women with PCO (PCO: 4230 ± 189 pg/mL [mean ± SEM], n = 188; non-PCO: 3498 ± 199 pg/mL, n = 168; P < 0.03), whereas BMP15 was lower in women with PCO (PCO: 431 ± 40 pg/mL, n = 125; non-PCO: 573 ± 55 pg/mL, n = 109; P = 0.10), leading to a significantly higher GDF9:BMP15 ratio in women with PCO (P < 0.01). Significant positive associations between BMP15 and AMH, activins, and inhibins in non-PCO women switched to negative associations in women with PCO. Conclusions Intrafollicular concentrations of GDF9 and BMP15 varied inversely in women with PCO reflecting an aberrant endocrine environment. An increased GDF9:BMP15 ratio may be a new biomarker for PCO

More pronounced salt dependence and higher reactivity for platination of the hairpin r(CGCGUUGUUCGCG) compared with d(CGCGTTGTTCGCG)

The DNA interference pathways exhibited by cisplatin and related anticancer active metal complexes have been extensively studied. Much less is known to what extent RNA interaction pathways may operate in parallel, and perhaps contribute to both antineoplastic activity and toxicity. The present study was designed with the aim of comparing the reactivity of two model systems comprising RNA and DNA hairpins, r(CGCGUUGUUCGCG) and d(CGC GTTGTTCGCG), towards a series of platinum(II) complexes. Three platinum complexes were used as metallation reagents; cis-[ptCl(NH3)(2)(OH2)](+) (1), cis-[PtCl(NH3)(C-C6H11NH2)(OH2)](+) (2), and trans[PtCl(NH3)(quinoline)(OH2)](+) (3). The reaction kinetics were studied at pH 6.0, 25 degrees C, and 1.0 mM < 1: 500 mM. For both types of nucleic acid targets, compound 3 was found to react about 1 order of magnitude more rapidly than compounds 1 and 2. Further, all platinum compounds exhibited a more pronounced salt dependence for the interaction with r(CGCGUUGUUCGCG). Chemical and enzymatic cleavage studies revealed similar interaction patterns with r(CGCGUUGUUCGCG) after long exposure times to 1 and 2. A substantial decrease of cleavage intensity was found at residues G4 and G7, indicative of bifunctional adduct formation. Circular dichroism studies showed that platinum adduct formation leads to a structural change of the ribonucleic acid. Thermal denaturation studies revealed platination to cause a decrease of the RNA melting temperatures by 5-10 degrees C. Our observations therefore suggest that RNA is a kinetically competitive target to DNA. Furthermore, platination causes destabilization of RNA structural elements, which may lead to deleterious intracellular effects on biologically relevant RNA targets