Search

New vaccine formulations that include novel strains of Mycoplasma hyopneumoniae and innovative adjuvants designed to induce cellular immunity could improve vaccine efficacy against this pathogen. The aim of this experimental study was to assess the efficacy of three experimental bacterin formulations based on M. hyopneumoniae field strain F7.2C which were able to induce cellular immunity. The formulations included a cationic liposome formulation with the Mincle receptor ligand trehalose 6,6-dibehenate (Lipo_DDA:TDB), a squalene-in-water emulsion with Toll-like receptor (TLR) ligands targeting TLR1/2, TLR7/8 and TLR9 (SWE_TLR), and a poly(lactic-co-glycolic acid) micro-particle formulation with the same TLR ligands (PLGA_TLR). Four groups of 12 M. hyopneumoniae-free piglets were primo- (day (D) 0; 39 days of age) and booster vaccinated (D14) intramuscularly with either one of the three experimental bacterin formulations or PBS. The pigs were endotracheally inoculated with a highly and low virulent M. hyopneumoniae strain on D28 and D29, respectively, and euthanized on D56. The main efficacy parameters were: respiratory disease score (RDS; daily), macroscopic lung lesion score (D56) and log copies M. hyopneumoniae DNA determined with qPCR on bronchoalveolar lavage (BAL) fluid (D42, D56). All formulations were able to reduce clinical symptoms, lung lesions and the M. hyopneumoniae DNA load in the lung, with formulation SWE_TLR being the most effective (RDSD28–D56 −61.90%, macroscopic lung lesions −88.38%, M. hyopneumoniae DNA load in BAL fluid (D42) −67.28%). Further experiments raised under field conditions are needed to confirm these results and to assess the effect of the vaccines on performance parameters.

The bone morphogenetic protein receptor II (BMPRII) signaling pathway is impaired in pulmonary arterial hypertension and mutations in the BMPR2 gene have been observed in both heritable and idiopathic pulmonary arterial hypertension. However, all BMPR2 mutation carriers do not develop pulmonary arterial hypertension, and inflammation could trigger the development of the disease in BMPR2 mutation carriers. Circulating levels and/or lung tissue expression of cytokines such as tumor necrosis factor-α or interleukin-18 are elevated in patients with pulmonary arterial hypertension and could be involved in the pathogenesis of pulmonary arterial hypertension. We consequently hypothesized that cytokines could trigger endothelial dysfunction in addition to impaired BMPRII signaling. Our aim was to determine whether impairment of BMPRII signaling might affect endothelium barrier function and adhesiveness to monocytes, in response to cytokines. BMPR2 was silenced in human lung microvascular endothelial cells (HLMVECs) using lentiviral vectors encoding microRNA-based hairpins. Effects of tumor necrosis factor-α and interleukin-18 on HLMVEC adhesiveness to the human monocyte cell line THP-1, adhesion molecule expression, endothelial barrier function and activation of P38MAPK were investigated in vitro. Stable BMPR2 silencing in HLMVECs resulted in impaired endothelial barrier function and constitutive activation of P38MAPK. Adhesiveness of BMPR2-silenced HLMVECs to THP-1 cells was enhanced by tumor necrosis factor-α and interleukin-18 through ICAM-1 adhesion molecule. Interestingly, tumor necrosis factor-α induced activation of P38MAPK and disrupted endothelial barrier function in BMPR2-silenced HLMVECs. Altogether, our findings showed that stable BMPR2 silencing resulted in impaired endothelial barrier function and activation of P38MAPK in HLMVECs. In BMPR2-silenced HLMVECs, cytokines enhanced adhesiveness capacities, activation of P38MAPK and impaired endothelial barrier function suggesting that cytokines could trigger the development of pulmonary arterial hypertension in a context of impaired BMPRII signaling pathway. ispartof: PULMONARY CIRCULATION vol:9 issue:4 ispartof: location:United States status: published

Bone morphogenetic protein receptor II (BMPRII) signaling pathway is impaired in pulmonary arterial hypertension (PAH) and mutations in the BMPR2 gene are associated with both heritable and idiopathic PAH. Endothelial dysfunction is a key process in initiation and progression of PAH pathogenesis. We aimed to investigate whether loss of BMPRII could affect endothelial function, including endothelial integrity and angiogenesis of pulmonary lung microvascular endothelial cells (HLMVEC). BMPR2 knockdown was performed in HLMVEC using lentiviral vectors encoding microRNA-based hairpins against BMPR2. HLMVEC lines were established and phenotyped. BMPRII protein expression and activation of BMPRII downstream effectors, SMAD and p38MAPK, were investigated by Western blotting. Effects of BMPR2 knockdown on endothelial integrity using a permeability assay and angiogenic capacities were further evaluated in vitro using migration and tube formation assays. Stable BMPR2 silencing did not affect HLMVEC phenotype, resulted in i) a 87% decrease in BMPRII protein expression, ii) a 50% decrease in SMAD protein activation and iii) a 48% increase in p38 MAPK phosphorylation. Interestingly, loss of BMPRII resulted in a 24% significant decrease in barrier function and a 29% decrease in migration capacity and impaired 3D-angiogenesis in vitro. Stable BMPR2 silencing in HLMVECs results in a shift in attenuated activation of the canonical SMAD signaling into enhanced activation of the non-canonical p38 MAPK signaling. In addition, impaired BMPRII signaling induced a loss of barrier function and decreased angiogenic capacities indicating the involvement of BMPRII in endothelial dysfunction in PAH.

This study aimed to investigate a possible involvement of mycotoxins in neonatal tail necrosis in piglets. Ten affected and 10 non-affected farms were selected. Sow feed samples were analysed for the presence of 23 mycotoxins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Blood plasma samples of sows and their piglets were analysed for the presence of deoxynivalenol (DON), de-epoxydeoxynivalenol, T-2 and HT-2 toxin, zearalenone, alfa-zearalenol, and beta-zearalenol, using LC-MS/MS. Additionally, high-resolution mass spectrometry (HRMS) was performed to detect DON-glucuronide (DON-Glca). There was a significant difference between case herds and control herds for mean DON concentrations in feed and sow plasma. For piglet samples, concentrations of DON were above the limit of quantification of 0.1 ng/ml in only 12 samples. Positive correlations were found between DON concentrations in sow feed and plasma of sows; DON concentration in sow feed and DON-Glca concentration in plasma of sows; and between DON and DON-Glca concentration in sow-plasma. In conclusion, high prevalence of DON in feed samples was found, with significantly higher concentrations in case herds, as well as the presence of DON and DON-Glca in sow plasma. Additional research is needed to identify risk factors, including within-herd factors, associated with neonatal tail necrosis in piglets.

International audience; The importance of diversity of Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is not yet fully known. This study investigated the genetic diversity of M. hyopneumoniae strains in ten pig herds, and assessed associations between the presence of different strains of M. hyopneumoniae and lung lesions at slaughter. Within each herd, three batches of slaughter pigs were investigated. At slaughter, from each batch, 20 post mortem bronchoalveolar lavage fluid samples were collected for multiple locus variable-number tandem repeat analysis (MLVA), and lung lesions (Mycoplasma-like lesions, fissures) were examined. Multivariable analyses including potential risk factors for respiratory disease were performed to assess associations between the number of different strains per batch (three categories: one strain, two–six strains, ≥seven strains), and the lung lesions as outcome variables. In total, 135 different M. hyopneumoniae strains were found. The mean (min.–max.) number of different strains per batch were 7 (1–13). Batches with two–six strains or more than six strains had more severe Mycoplasma-like lesions (P = 0.064 and P = 0.012, respectively), a higher prevalence of pneumonia [odds ratio (OR): 1.30, P = 0.33 and OR: 2.08, P = 0.012, respectively], and fissures (OR = 1.35, P = 0.094 and OR = 1.70, P = 0.007, respectively) compared to batches with only one strain. In conclusion, many different M. hyopneumoniae strains were found, and batches of slaughter pigs with different M. hyopneumoniae strains had a higher prevalence and severity of Mycoplasma-like lung lesions at slaughter, implying that reducing the number of different strains may lead to less lung lesions at slaughter and better respiratory health of the pigs.

Background: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary agent of enzootic pneumonia in pigs. Pigs are often infected with different M. hyopneumoniae strains. This study assessed the efficacy of vaccination against experimental infection with two genetically different M. hyopneumoniae strains in weaned piglets. At 33 days of age (D0), 45 M. hyopneumoniae-free piglets were randomly assigned to three different groups: 1) negative control group (NCG; n = 5): not vaccinated, not infected, 2) positive control group (PCG; n = 20): not vaccinated, infected, and 3) vaccination group (VG; n = 20): single vaccination with an inactivated whole-cell M. hyopneumoniae vaccine (Hyogen (R), Ceva) (D1), infected. The PCG and VG were endotracheally inoculated with 7 x 10(7) CCU in 7 ml of the highly virulent M. hyopneumoniae strain F7.2C (D24) and 7 x 10(7) CCU in 7 ml low virulent strain F1.12A (D25). A respiratory disease score (RDS) was assessed from D24 until D53. At D53 (euthanasia), macroscopic lung lesions (MLL) were scored, log copies of M. hyopneumoniae DNA (qPCR) and IL-1 and IL-6-concentrations (ELISA) on bronchoalveolar lavage fluid were determined. Results: The RDS and MLL at euthanasia were respectively 0, 1.20 and 0.55 (P < 0.001) and 0, 7.56 and 0.68 (P < 0.001) for NCG, PCG and VG, respectively. The qPCR results for PCG and VG were 3.99 and 1.78 log copies (P < 0.001), respectively, with a significant difference between PCG and VG. The IL-1 and IL-6 results at euthanasia for NCG, PCG and VG were 17.61, 1283.39 and 53.04 pg/ml (P < 0.001) and 148.10, 493.35 and 259.80 pg/ml (P = 0.004), respectively with a significant difference between PCG and VG. Conclusions: Vaccination with Hyogen r in pigs was efficacious against an experimental challenge with both a low and highly virulent M. hyopneumoniae strain as the vaccinated pigs coughed significantly less, and showed significantly less lung lesions compared to the non-vaccinated challenged pigs: the vaccinated animals showed a 52.9% lower RDS and 91.0% lower MLL compared to the PCG. In the bronchoalveolar lavage fluid collected at the necropsy of the vaccinated pigs, a significantly lower amount of M. hyopneumoniae-DNA and a significantly lower IL-1 and IL-6 concentration was found compared to the pigs of the PCG.

This study assessed the efficacy of two different Mycoplasma hyopneumoniae vaccination programmes in relation to the time of weaning. Eight hundred and twenty-eight piglets were randomly divided into three groups: group V1 was vaccinated three days before weaning, group V2 at weaning (21 days of age) and group NV was left non-vaccinated. Vaccinations were performed using Ingelvac MycoFLEX. After the nursery period, 306 pigs were allocated to fattening unit (F1) and 501 pigs to a second unit (F2). Efficacy was evaluated using performance parameters and pneumonia lesions at slaughter. Statistically significant differences were obtained in F2 where group V1 had a higher average daily weight gain compared to groups V2 and NV for the entire study period (17 and 18 g/day, respectively) and the fattening period (26 and 36 g/day, respectively) (P0.05). Between groups, there were no statistically significant differences for the average lung lesion scores (V1=3.44; V2=4.61; NV=4.55, P>0.05) and the prevalence of pneumonia (V1=35.0 per cent; V2=38.0 per cent; NV=41.4 per cent, P>0.05). Overall, vaccination against M hyopneumoniae before weaning provided numerically better performance than vaccination at weaning, but did not reach statistical significance. An influenza outbreak in F1 and the presence of coexisting mixed respiratory infections in both F1 and F2 could have possibly influenced the performance of both vaccinated groups across all measured parameters.

A recombinant chimaeric protein containing three Mycoplasma hyopneumoniae antigens (C-terminal portion of P97, heat shock protein P42, and NrdF) fused to an adjuvant, the B subunit of heat-labile enterotoxin of Escherichia coli (LTB), was used to immunize pigs against enzootic pneumonia. The systemic and local immune responses, as well as the efficacy of the chimaeric protein in inducing protection against experimental M. hyopneumoniae infection were evaluated. In total, 60 male piglets, purchased from a M. hyopneumoniae-free herd, at 4 weeks of age were randomly allocated to six different experimental groups of 10 animals each: recombinant chimaeric protein by intramuscular (IM) (1) or intranasal (IN) (2) administration, commercial bacterin by IM administration (3), and the adjuvant LTB by IM (4, control group A) or IN (5, control group B) administration. All groups were immunized at 24 and 38 days of age and challenged at 52 days of age. The sixth group that was not challenged was used as the negative control (IN [n=5] or IM [n=5] administration of the LTB adjuvant). Compared with the non-challenged group, administration of the chimaeric protein induced significant (P

This study investigated Mycoplasma hyopneumoniae colonisation and lung lesions at slaughter in pigs from vaccinated (V) and non-vaccinated (NV) sows, in two herds (A and B). In each herd, two sow batches were V against M. hyopneumoniae with a commercial bacterin at six and three weeks before farrowing and two sow batches remained NV. From each sow batch, laryngeal swabs were collected from the litters of five primiparous sows at weaning and seven days post-weaning. All samples were tested for M. hyopneumoniae by nested PCR. In total, 488 piglets were sampled. At slaughter, the extent of Mycoplasma-like pneumonia lesions (lung lesion score (LLS)) was assessed. The colonisation rates with M. hyopneumoniae at weaning and seven days post-weaning were (V-A=14.2, NV-A=20.0 (P=0.225); V-B=0.9, NV-B=0.8 (P=0.948)) and (V-A=0.8, NV-A=7.0 (P=0.039); V-B=1.8, NV-B=2.5 (P=0.738)), respectively. The average LLS (in per cent) was V-A=15.5, NV-A=26.4 (P=0.021); V-B=9.7, NV-B=8.4 (P=0.541). In conclusion, in herd A, with a substantially higher level of piglet colonisation at weaning than herd B, offspring from V sows had a significantly lower colonisation rate seven days post-weaning and a significantly lower LLS at slaughter compared with the offspring of the NV sows. This implies that sow vaccination might be useful for control of M. hyopneumoniae infections, although significant results may not be achieved at all times (such as in herd B).

Mutations in the bone morphogenetic protein receptor 2 ( BMPR2 ) gene are associated with heritable pulmonary arterial hypertension (PAH). In addition, BMPRII signalling is impaired in idiopathic PAH. Since BMPR2 mutation carriers do not all develop PAH, inflammatory mediators could trigger the disease in BMPR2 mutation carriers. Since circulating levels and pulmonary tissue expression of interleukin-18 (IL18) are elevated in PAH, we hypothesized that IL18 could contribute to PAH pathogenesis, in addition to impaired BMPRII signalling. Lentiviral vectors encoding microRNA 30-based knockdown hairpins derived from BMPR2 siRNAs were generated and transduced in human lung microvascular endothelial cells (HLMVEC) to mimic impaired BMPR2 signalling in vitro. Effects of IL-18 on i) adhesion of HLMVEC to human monocytic cell line THP-1, endothelial integrity, adhesion molecule expression and activation of p38 MAP kinase and SMAD proteins were investigated. Knockdown of BMPR2 in HLMVEC resulted in decreased activation of SMAD proteins, increased activation of p38 MAP kinase and endothelium permeability. In BMPR2 knocked-down HLMVEC, IL18 and TNFα increased adhesion to monocytes by 35% and 77%, respectively and activated p38 MAP kinase. Interestingly, TNFα induced mRNA IL18 receptor expression in BMPR2 knocked-down HLMVEC. To conclude, in BMPR2 knocked-down HLMVEC, IL18 induced adhesion and p38 MAP kinase activation, suggesting that IL-18 could trigger PAH pathogenesis, in addition to BMPR2 impaired signalling.

Background: Commercial bacterins are widely used at weaning to control Mycoplasma hyopneumoniae infections in pigs. However, it is not known whether the efficacy of vaccinating against M. hyopneumoniae can be influenced by the weaning process when vaccination is applied at the day of weaning. The present study assessed the efficacy of a single M. hyopneumoniae vaccination (Ingelvac MycoFLEX (R)) three days before weaning (V1) or at weaning (V2) against experimental challenge infection. Four weeks after vaccination, groups V1 and V2 (n = 20 pigs each) and a non-vaccinated, positive control group (PCG) (n = 20) were endotracheally inoculated with a virulent M. hyopneumoniae field strain. Five pigs were used as a negative control group. All pigs were euthanized 5 weeks after challenge. The main parameters investigated included macroscopic and histopathological lung lesions at necropsy, immunofluorescence (IF) staining and quantitative real-time PCR (qPCR) on broncho-alveolar lavage (BAL) fluid for quantifying M. hyopneumoniae. Results: The average macroscopic lung lesion scores in groups V1, V2 and PCG were 0.54, 0.88 and 1.04, respectively (P > 0.05). The average lymphohistiocytic infiltration scores in groups V1, V2 and PCG were 2.95, 3.16 and 3.61, respectively (P < 0.05). The average IF scores were: V1 = 1.13, V2 = 1.19 and PCG = 1.25 (P > 0.05), the qPCR values were: V1 = 10(2.94), V2= 10(2.76) and PCG = 10(3.23) (P > 0.05). All pigs of the negative control group remained negative throughout the study. Conclusions: Both vaccinated groups had lower numbers of macroscopic and histopathological lung lesions, and lower numbers of M. hyopneumoniae organisms in the BAL fluid compared to the PCG. However, no firm conclusions could be made on whether weaning negatively influences the efficacy of M. hyopneumoniae vaccination, since significant differences between the treatment groups were only obtained for the histopathological lung lesions. This could be attributed to the fact that milder macroscopic lung lesions were produced in the inoculated pigs, when compared to previous trials conducted by the same group. Further research under field conditions is warranted to assess possible differences between the two vaccination strategies.

Dendritic cells (DCs) are an attractive tool for immunomodulation, targeting mature DCs (mDCs) for immunization or immature/semimature DCs (iDCs) for tolerization. Therefore, introducing antigens into DCs has become a prime topic in various immunological disciplines. Numerous studies have shown that lentiviruses are an efficient vehicle for this purpose. This study evaluates the effects of lentiviral transduction on iDC activation. Immature DCs are efficiently transduced with increasing doses of lentivirus without affecting cell viability. Transduction at low multiplicities of infection (MOIs) did not result in phenotypical or functional maturation. Higher doses of lentivirus, however, resulted in upregulation of adhesion, costimulatory, and HLA molecules, as well as in increased allostimulatory capacity and secretion of interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha. Production of IL-12 p70, IL-10, and interferon-alpha was observed only at extremely high doses. Protein kinase R phosphorylation on transduction at an MOI of 150 was demonstrated by Western blotting. A Toll-like receptor (TLR)-driven luciferase reporter assay showed dose-dependent activation of TLR2, TLR3, and TLR8, which was independent of the pseudotype, production, or transduction protocol and was abrogated on heat inactivation. These data show that lentiviral vectors provide not only the antigen but also appropriate activation signals to iDCs, favoring their use for immunotherapy and vaccine development.

BACKGROUND: Alpha-synuclein (α-SYN) aggregates represent a key feature of Parkinson's disease, but the exact relationship between α-SYN aggregation and neurodegeneration remains incompletely understood. Therefore, the availability of a cellular assay that allows medium-throughput analysis of α-SYN-linked pathology will be of great value for studying the aggregation process and for advancing α-SYN-based therapies. NEW METHOD: Here we describe a high-content neuronal cell assay that simultaneously measures oxidative stress-induced α-SYN aggregation and apoptosis. RESULTS: We optimized an automated and reproducible assay to quantify both α-SYN aggregation and cell death in human SH-SY5Y neuroblastoma cells. COMPARISON WITH EXISTING METHODS: Quantification of α-SYN aggregates in cells has typically relied on manual imaging and counting or cell-free assays, which are time consuming and do not allow a concurrent analysis of cell viability. Our high-content analysis method for quantification of α-SYN aggregation allows simultaneous measurements of multiple cell parameters at a single-cell level in a fast, objective and automated manner. CONCLUSIONS: The presented analysis approach offers a rapid, objective and multiparametric approach for the screening of compounds and genes that might alter α-SYN aggregation and/or toxicity.

Viral vector-mediated expression of genes (e.g., coding for opsins and designer receptors) has grown increasingly popular. Cell-type specific expression is achieved by altering viral vector tropism through crosspackaging or by cell-specific promoters driving gene expression. Detailed information about transduction properties of most recombinant adeno-associated viral vector (rAAV) serotypes in macaque cortex is gradually becoming available. Here, we compare transduction efficiencies and expression patterns of reporter genes in two macaque neocortical areas employing different rAAV serotypes and promoters. A short version of the calmodulin-kinase-II (CaMKIIα0.4) promoter resulted in reporter gene expression in cortical neurons for all tested rAAVs, albeit with different efficiencies for spread: rAAV2/5>>rAAV2/7>rAAV2/8>rAAV2/9>>rAAV2/1 and proportion of transduced cells: rAAV2/1>rAAV2/5>rAAV2/7=rAAV2/9>rAAV2/8. In contrast to rodent studies, the cytomegalovirus (CMV) promoter appeared least efficient in macaque cortex. The human synapsin-1 promoter preceded by the CMV enhancer (enhSyn1) produced homogeneous reporter gene expression across all layers, while two variants of the CaMKIIα promoter resulted in different laminar transduction patterns and cell specificities. Finally, differences in expression patterns were observed when the same viral vector was injected in two neocortical areas. Our results corroborate previous findings that reporter-gene expression patterns and efficiency of rAAV transduction depend on serotype, promoter, cortical layer, and area. ispartof: Neurophotonics vol:2 issue:3 pages:031209- ispartof: location:United States status: published

Dendritic cells (DC) are professional antigen-presenting cells that are used in vaccine approaches to cancer. Classically, mature monocyte-derived DC are generated in vitro in the presence of interleukin (IL)-4, granulocyte macrophage-colony stimulating factor, and inflammatory cytokines (G4-DC). Recently, it has been described that DC can also be generated in the presence of IL-3 and interferon (IFN)-β and that these DC are efficiently matured using polyriboinosinic polyribocytidylic acid (I3-DC). In this study, a series of in vitro experiments was performed to compare side-by-side I3-DC and G4-DC as vaccine adjuvants. Phenotypic characterization of the DC revealed differences in the expression of the monocyte marker CD14 and the maturation marker CD83. Low expression of CD14 and high expression of CD83 characterized G4-DC, whereas I3-DC displayed intermediate expression of CD14 and CD83. Both types of DC were as potent in the induction of allogeneic T cell proliferation. Upon CD40 ligation, G4-DC produced lower amounts of IFN-α and pulmonary and activation-regulated chemokine, similar amounts of IL-6, macrophage-inflammatory protein (MIP)-1α, and MIP-1β, and higher amounts of IL-12 p70, tumor necrosis factor α, and MIP-3β than I3-DC. We further evaluated whether the DC could be frozen/thawed without loss of cell number, viability, phenotype, and function. After freezing/thawing, 56.0% ± 9.0% of I3-DC and 77.0% ± 3.0% of G4-DC (n=9) were recovered as viable cells, displaying the same phenotype as their fresh counterparts. Finally, in vitro stimulations showed that fresh and frozen peptide-loaded I3-DC are more potent inducers of Melan-A-specific CD8+ T cell responses than G4-DC. The antigen-specific T cells were functional as shown in cytotoxicity and IFN-γ secretion assay.

An optimal anticancer vaccine probably requires the cooperation of both CD4+ Th cells and CD8+ CTLs. A promising tool in cancer immunotherapy is, therefore, the genetic modification of dendritic cells (DCs) by introducing the coding region of a tumor Ag, of which the antigenic peptides will be presented in both HLA class I and class II molecules. This can be achieved by linking the tumor Ag to the HLA class II-targeting sequence of an endosomal or lysosomal protein. In this study we compared the efficiency of the targeting signals of invariant chain, lysosome-associated membrane protein-1 (LAMP1) and DC-LAMP. Human DCs were electroporated before or after maturation with mRNA encoding unmodified enhanced green fluorescent protein (eGFP) or eGFP linked to various targeting signals. The lysosomal degradation inhibitor chloroquine was added, and eGFP expression was evaluated at different time points after electroporation. DCs were also electroporated with unmodified MAGE-A3 or MAGE-A3 linked to the targeting signals, and the presentation of MAGE-A3-derived epitopes in the context of HLA class I and class II molecules was investigated. Our data suggest that proteins linked to the different targeting signals are targeted to the lysosomes and are indeed presented in the context of HLA class I and class II molecules, but with different efficiencies. Proteins linked to the LAMP1 or DC-LAMP signal are more efficiently presented than proteins linked to the invariant chain-targeting signal. Furthermore, DCs electroporated after maturation are more efficient in Ag presentation than DCs electroporated before maturation.

The main objective of the study was to assess particulate matter (PM) exposure levels for both the farmer and the veterinarian during different operational tasks in pig-fattening houses, and to estimate their exposure levels on a daily working basis (time-weighted average (TWA)). The measured PM fractions were: inhalable and respirable PM, PM10, PM2.5 and PM1. The effects of pig age, pen floor type (conventional or low emission surface) and cleaning of the pens on the personal PM exposure were also investigated. Indoor concentrations of NH[sub]3[/sub], CH[sub]4[/sub], and CO[sub]2[/sub] were additionally measured during some operational tasks. The results showed that personal exposure levels can become extremely high during some operational tasks performed by the farmer or veterinarian. The highest concentration levels were observed during feed shovelling and blood sampling, the lowest during the weighing of the pigs. For the farmer, the estimated TWA exposure levels of inhalable and respirable PM were 6.0 and 0.29 mg m[sup] -3[/sup] , respectively. These exposure levels for the veterinarian were, respectively, 10.6 and 0.74 mg m[sup] -3[/sup] . The PM concentration levels were mainly determined by the performed operational tasks. There was no significant effect of pig age, pen floor type, nor cleaning of the pens on the personal exposure levels.

The present study investigated the simultaneous influence of particulate matter (PM10) and ammonia (NH3) on performance, lung lesions and the presence of Mycoplasma hyopneumoniae (M. hyopneumoniae) in finishing pigs. A pig herd experiencing clinical problems of M. hyopneumoniae infections was selected. In total, 1095 finishing pigs of two replicates in eight compartments each were investigated during the entire finishing period (FP). Indoor PM10 and NH3 were measured at regular intervals during the FP with two Grimm spectrometers and two Graywolf Particle Counters (PM10) and an Innova photoacoustic gas monitor (NH3). Average daily weight gain (ADG) and mortality were calculated and associated with PM10 and NH3 during the FP. Nasal swabs (10 pigs/compartment) were collected one week prior to slaughter to detect DNA of M. hyopneumoniae with nested PCR (nPCR). The prevalence and extent of pneumonia lesions, and prevalence of fissures and pleurisy were examined at slaughter (29 weeks). The results from the nasal swabs and lung lesions were associated with PM10 and NH3 during the FP and the second half of the FP. In the univariable model, increasing PM10 concentrations resulted in a higher odds of pneumonia lesions (second half of the FP: OR=8.72; P=0.015), more severe pneumonia lesions (FP: P=0.04, second half of the FP: P=0.009), a higher odds of pleurisy lesions (FP: OR=20.91; P

Antigen-loaded dendritic cells (DCs) have been intensively investigated as potential cellular antitumor vaccines. Several recent reports have indicated that loading DCs with whole tumor derived mRNA or defined tumor-antigen-encoding mRNA represents an effective nonviral strategy to stimulate T cell responses both for in vitro and in vivo models. Here, we describe the electroporation method as a tool for introducing in vitro transcribed capped mRNA into human DCs for tumor vaccination. We use MART-1/Melan-A as a model tumor-associated antigen for the generation of a DC-based vaccine against melanoma cancer. In addition to efficient antigen loading, it is important to obtain a maximal number of potent antigen-presenting cells. Another prerequisite for the development of a DC-based cancer vaccine is to obtain mature DCs. In this chapter, we describe the basic techniques required for the successful genetic modification of DCs by using the mRNA electroporation method.

Antigen-loaded dendritic cells (DCs) have been intensively investigated as potential cellular antitumor vaccines. Several recent reports have indicated that loading DCs with whole tumor derived mRNA or defined tumor-antigen-encoding mRNA represents an effective nonviral strategy to stimulate T cell responses both for in vitro and in vivo models. Here, we describe the electroporation method as a tool for introducing in vitro transcribed capped mRNA into human DCs for tumor vaccination. We use MART-1/Melan-A as a model tumor-associated antigen for the generation of a DC-based vaccine against melanoma cancer. In addition to efficient antigen loading, it is important to obtain a maximal number of potent antigen-presenting cells. Another prerequisite for the development of a DC-based cancer vaccine is to obtain mature DCs. In this chapter, we describe the basic techniques required for the successful genetic modification of DCs by using the mRNA electroporation method.

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutic strategies. One of the approaches that has gained prominence in recent years is therapeutic vaccination. We decided to assess the capacity of mature dendritic cells, derived from blood monocytes of HIV-1 infected patients, to generate functional T-cell responses. For this purpose, we constructed a chimeric mRNA encoding the proteins Tat, Rev and Nef. The TaReNef encoding information was linked to the HLA class II-targeting sequence of DC-LAMP. Broadly directed HIV-specific CD4(+) and CD8(+) cytotoxic T cells exhibiting a poly-functional cytokine secretion pattern were generated by co-culturing with autologous chimeric mRNA electroporated dendritic cells. Thus, administration of ex vivo generated dendritic cells expressing the early proteins Tat, Rev and Nef might offer a promising approach for therapeutic vaccination in HIV-1 infection. (C) 2008 Elsevier Ltd. All rights reserved.

Until now, studies utilizing mRNA electroporation as a tool for the delivery of tumor antigens to human monocyte-derived dendritic cells (DC) have focused on DC electroporated in an immature state. Immature DC are considered to be specialized in antigen capture and processing, whereas mature DC present antigen and have an increased T-cell stimulatory capacity. Therefore, the consensus has been to electroporate DC before maturation. We show that the transfection efficiency of DC electroporated either before or after maturation was similarly high. Both immature and mature electroporated DC, matured in the presence of an inflammatory cytokine cocktail, expressed mature DC surface markers and preserved their capacity to secrete cytokines and chemokines upon CD40 ligation. In addition, both immature and mature DC can be efficiently cryopreserved before or after electroporation without deleterious effects on viability, phenotype or T-cell stimulatory capacity including in vitro antigen-specific T-cell activation. However, DC electroporated after maturation are more efficient in in vitro migration assays and at least as effective in antigen presentation as DC electroporated before maturation. These results are important for vaccination strategies where an optimal antigen presentation by DC after migration to the lymphoid organs is crucial.

The use of tumor antigen-loaded dendritic cells (DC) is one of the most promising approaches to inducing a tumor-specific immune response. We compared electroporation of mRNA to lentiviral transduction for the delivery of tumor antigens to human monocyte-derived and murine bone marrow-derived DC. Both lentiviral transduction and mRNA electroporation induced eGFP expression in on average 81% of human DC. For murine DC, eGFP mRNA electroporation (62%) proved to be more efficient than lentiviral transduction (47%). When we used tNGFR as a transgene we observed lentiviral pseudotransduction that overestimated lentiviral efficiency. Neither gene transfer method had an adverse effect on viability, phenotype, or allostimulatory capacity of either human or murine DC. Yet, the mRNA-electroporated DC showed a reduced production of IL-12p70 compared to their lentivirally transduced and unmodified counterparts. Human Ii80MAGE-A3-modified DC and murine Ii80tOVA-modified DC were able to present antigenic epitopes in the context of MHC class I and class II. Both types of modified murine DC were able to induce OVA-specific cytotoxic T cells in vivo; however, the mRNA-electroporated DC were less potent. Our data indicate that this may be related to their impaired IL-12 production.

We previously described mRNA electroporation as an efficient gene delivery method to introduce tumor-antigens (Ag) into murine immature dendritic cells (DC). Here, we further optimize the protocol and evaluate the capacity of mRNA-electroporated DC as a vaccine for immunotherapy. First, the early DC maturation kinetics and the effect of different lipopolysaccharide incubation periods on the phenotypic maturation profile of DC are determined. Next, we show that either immature or mature DC are equally well electroporated and express and present the transgene at a comparable level after electroporation. We point out that the mRNA electroporation results in a negative effect on the interleukin (IL)-12p70, IL-6, and tumor necrosis factor-α secretion after maturation. Nevertheless, mRNA-electroporated DC induce an effective cytotoxic T lymphocyte (CTL) response in vivo . Mature electroporated DC are significantly more potent in eliciting an Ag-specific CD8 + CTL response compared to their immature electroporated counterparts. In addition, a significant improvement in CTL response is obtained both in the primary and in the memory effector phases when CD4 + CD25 + regulatory T cells (Treg) are depleted in vivo prior to immunization. These findings are further substantiated in tumor protection experiments and hold convincing evidence for the merit of Treg cell depletion prior to immunization with mRNA-electroporated DC.

For the induction of an optimal immune response against cancer or infections not only CD8(+) CTLs but also CD4(+) T helper cells must be induced, in particular IFN-gamma-secreting type 1 T helper cells. Several strategies have been explored to target tumor-associated antigens to the HLA class II processing compartments. We engineered a genetic construct encoding an invariant chain (Ii) protein where the CLIP region has been replaced by sequences encoding HLA class II-restricted MAGE-A3 epitopes. Monocyte-derived dendritic cells (DCs) were electroporated with in vitro transcribed mRNA encoding a modified Ii protein containing the HLA-DP4-restricted MAGE-A3 epitope. The capacity of these electroporated DCs to stimulate a MAGE-A3-specific T-cell clone was compared at different stages of DC maturation with the T-cell stimulatory capacity of DCs pulsed with the synthetic peptide. After electroporation, the T-cell stimulatory capacity was shown to be high and long lasting, whereas the stimulatory capacity of peptide-pulsed DCs decreased rapidly. Upon coculture with epitope-specific T cells, electroporated immature DCs expressed enhanced levels of costimulatory molecules, HLA class II molecules, and CD83, suggesting the induction of maturation. The electroporated DCs can be frozen and thawed without losing their capability to stimulate the specific T-cell clone in vitro, and they are able to stimulate unprimed CD4(+) T cells specific to the HLA-DP4-restricted MAGE-A3 epitope in vitro. Similar results were obtained with a recombinant Ii containing the MAGE-A3 epitope presented in the context of HLA-DR13.

For the induction of an optimal immune response against cancer or infections not only CD8(+) CTLs but also CD4(+) T helper cells must be induced, in particular IFN-gamma-secreting type 1 T helper cells. Several strategies have been explored to target tumor-associated antigens to the HLA class II processing compartments. We engineered a genetic construct encoding an invariant chain (Ii) protein where the CLIP region has been replaced by sequences encoding HLA class II-restricted MAGE-A3 epitopes. Monocyte-derived dendritic cells (DCs) were electroporated with in vitro transcribed mRNA encoding a modified Ii protein containing the HLA-DP4-restricted MAGE-A3 epitope. The capacity of these electroporated DCs to stimulate a MAGE-A3-specific T-cell clone was compared at different stages of DC maturation with the T-cell stimulatory capacity of DCs pulsed with the synthetic peptide. After electroporation, the T-cell stimulatory capacity was shown to be high and long lasting, whereas the stimulatory capacity of peptide-pulsed DCs decreased rapidly. Upon coculture with epitope-specific T cells, electroporated immature DCs expressed enhanced levels of costimulatory molecules, HLA class II molecules, and CD83, suggesting the induction of maturation. The electroporated DCs can be frozen and thawed without losing their capability to stimulate the specific T-cell clone in vitro, and they are able to stimulate unprimed CD4(+) T cells specific to the HLA-DP4-restricted MAGE-A3 epitope in vitro. Similar results were obtained with a recombinant Ii containing the MAGE-A3 epitope presented in the context of HLA-DR13.

Genetically modified dendritic cells (DC) constitute a promising approach in cancer immunotherapy. Viral gene delivery systems have been shown to be very efficient strategies, but safety concerns for their clinical use in immunotherapy remain an important issue. Recently, the technique of mRNA electroporation was described as a very efficient tool for the genetic modification of human monocyte-derived DC. Here, we show that transgene expression can be modulated by varying the amount of mRNA used for electroporation. We document that CD40 ligation leads to a significant production of IL-12 by the electroporated DC, although the level of IL-12 production is somewhat lower than for non- or mock-electroporated DC. Furthermore, we show that the electroporated DC can be frozen and thawed without loss of viability or function and that Influenza virus Matrix Protein 1 mRNA electroporated DC are capable of inducing a memory cytotoxic T lymphocyte response and are more potent in doing so than mRNA-pulsed DC. Similar results were obtained with MelanA/MART-1 mRNA electroporated DC. These results clearly indicate that mRNA-electroporated DC represent powerful candidates for use as tumor vaccines and could constitute an improvement compared with vaccines using peptide-pulsed DC.

The efficacy of an Actinobacillus pleuropneumoniae subunit vaccine based on ApxIA, ApxIIA, ApxIIIA and OMP-2 (Porcilis App, MSD) was investigated in two farrow-to-finish pig herds (A and B) affected by chronic pleurisy. In total, 1161 pigs were included. At three weeks of age, the pigs were randomly allocated to non-vaccinated control (NV; n=580) and vaccinated (V; n=581) groups. At 6 and 10 weeks of age, pigs were injected with Porcilis-APP (V group) or adjuvant (NV group). At slaughter (26 weeks), pleurisy and pneumonia lesions were assessed. All pigs were weighed individually at 6 and 26 weeks of age, and average daily weight gain (ADG; g/pig/day) was calculated. Mortality and days of additional treatment (DAT) were registered during the whole experiment. Data were analysed using binary logistic regression or analysis of variance for proportions or continuous variables, respectively. The prevalence of pleurisy and pneumonia was (NV-A=19.3, V-A=7.9, (P=0.000); NV-B=17.9, V-B=0.7, (P=0.000)) and (NV-A=42.4, V-A=21.2, (P=0.000); NV-B=46.7, V-B=19.0, (P=0.000)), respectively. The ADG was NV-A=632±157, V-A=647±91, (P=0.162); NV-B=660±115, V-B=670±82, (P=0.232). The mortality during the experiment was NV-A=5.7, V-A=1.8, (P=0.015); NV-B=2.3, V-B=1.0, (P=0.170) per cent. The DAT was: NV-A=15.04±1.41, V-A=14.95±0.67, (P=0.010); NV-B=21.68±2.43, V-B=16.99±0.62, (P=0.000). The present study showed a significant reduction of the prevalence of pleurisy and pneumonia, and antimicrobial use in V pigs from both herds, and in mortality in V pigs from one herd.

The use of tumor antigen-loaded dendritic cells (DC) is one of the most promising approaches to inducing a tumor-specific immune response. We compared electroporation of mRNA to lentiviral transduction for the delivery of tumor antigens to human monocyte-derived and murine bone marrow-derived DC. Both lentiviral transduction and mRNA electroporation induced eGFP expression in on average 81% of human DC. For murine DC, eGFP mRNA electroporation (62%) proved to be more efficient than lentiviral transduction (47%). When we used tNGFR as a transgene we observed lentiviral pseudotransduction that overestimated lentiviral efficiency. Neither gene transfer method had an adverse effect on viability, phenotype, or allostimulatory capacity of either human or murine DC. Yet, the mRNA-electroporated DC showed a reduced production of IL-12p70 compared to their lentivirally transduced and unmodified counterparts. Human Ii80MAGE-A3-modified DC and murine Ii80tOVA-modified DC were able to present antigenic epitopes in the context of MHC class I and class II. Both types of modified murine DC were able to induce OVA-specific cytotoxic T cells in vivo; however, the mRNA-electroporated DC were less potent. Our data indicate that this may be related to their impaired IL-12 production.

Genetically modified dendritic cells (DC) constitute a promising approach in cancer immunotherapy. Viral gene delivery systems have been shown to be very efficient strategies, but safety concerns for their clinical use in immunotherapy remain an important issue. Recently, the technique of mRNA electroporation was described as a very efficient tool for the genetic modification of human monocyte-derived DC. Here, we show that transgene expression can be modulated by varying the amount of mRNA used for electroporation. We document that CD40 ligation leads to a significant production of IL-12 by the electroporated DC, although the level of IL-12 production is somewhat lower than for non- or mock-electroporated DC. Furthermore, we show that the electroporated DC can be frozen and thawed without loss of viability or function and that Influenza virus Matrix Protein 1 mRNA electroporated DC are capable of inducing a memory cytotoxic T lymphocyte response and are more potent in doing so than mRNA-pulsed DC. Similar results were obtained with MelanA/MART-1 mRNA electroporated DC. These results clearly indicate that mRNA-electroporated DC represent powerful candidates for use as tumor vaccines and could constitute an improvement compared with vaccines using peptide-pulsed DC.

In this study, we compared dendritic cells (DCs) differentiated from positively selected monocytes (CD14-DCs) to DCs differentiated from adherence-selected monocytes (adh-DCs) with emphasis on lentiviral transduction. Using a second-generation, triple-helix containing, self-inactivating lentiviral vector at a multiplicity of infection (MOI) of 15, we observed enhanced transduction of CD14-DCs (72.8 +/- 5.3%, mean fluorescence intensity [MFI] = 166 +/- 76) compared to adh-DCs (32.3 +/- 13.1%, MFI = 119 +/- 76, n = 5). More importantly, the efficiency to transduce adh-DCs was significantly increased when monocytes were incubated with antiCD14 antibody coupled beads, anti-CD14 antibodies, or lipopolysaccharide (LPS), reaching transduction efficiencies up to 86.6%, 53.3%, and 80.9%, respectively. We showed that this enhanced transduction was correlated to an activation of the monocytes, characterized by the up regulation of the cytokines interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha and the de novo synthesis of IL-6 and IL-10. However, the enhanced transduction of immature CD14-DCs was not correlated with a progression in the cell cycle from G(0) to G(1). We further showed that CD14-DCs were phenotypically comparable to adh-DCs. Functional analysis revealed that there were no differences in allostimulatory capacity, production of IL-12 p70 on CD40 ligation or expression of IL-1beta, IL-6, IL-8, IL-10, IL-12, and TNF-alpha as evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Finally, we showed that lentivirally transduced CD14-DCs were equally capable as adh-DCs in stimulating MAGE-A3 antigen-specific CD4(+) and CD8(+) T cells in vitro.