Evaluation of a heating protocol and stocking density impact on heatstressed fattening pigs.

As climate change intensifies, heat stress mitigation for pigs becomes more important. Trials involving induced heat waves are useful to test several measures (e.g. reduced stocking density) at a faster rate, but only when accurately evaluated and validated. In the present study, we investigated the suitability of an artificial heating protocol at different pig weights (experiment 1). The impact of different stocking densities on fattening pigs during an artificial heat wave (experiment 2) was also investigated. Experiment 1: Forty 20-week-old pigs weighing 96.5 ± 7.3 kg (W100) and forty 17-week-old pigs weighing 72.7 ± 9.9 kg (W70) were housed in two compartments. An artificial heat wave (heat load) was induced for 3 days. During 3-day periods before, during and after the heat load, physiological parameters (respiration rate (RR), rectal temperature (Trectal), skin temperature (Tskin) and behavior) were measured and average daily feed intake was observed. Ambient temperature, relative humidity and temperature-humidity index (THI) were monitored. Experiment 2: A total of 150 fattening pigs were randomly divided into three treatment groups: SD1.3 (1.3 m2/pig), SD1.0 (1.0 m2/pig) and SD0.8 (0.8 m2/pig). All pens had a total pen surface of 4.88 m2, corresponding with 4, 5 and 6 fattening pigs in the SD1.3, SD1.0 and SD0.8 groups, respectively. The heat load was induced for 7 days on week 21. Respiration rate and Trectal were observed as in experiment 1. Average daily gain and average daily feed intake were also noted. During the heat load, THI reached ≥ 75 (78.4 (experiment 1) and 78.6 (experiment 2)), even when relative humidity decreased to ± 45%. Every physiological parameter showed significant increases during the heat load. The prolonged heating protocol in experiment 2 also provoked significant decreases in average daily feed intake (15%) and average daily gain (19%) for all groups. Weight within the studied range of 70-100 kg did not have a significant impact on any of the parameters. However, Tskin was affected by both weight and heat load (P < 0.05), where Tskin from W100 was always lower in comparison to W70. In addition, we found that 0.8 m2/pig doubled the increase of Trectal during the heat load, namely SD0.8 (0.22 °C) compared to SD1.0 (0.12 °C) (P = 0.033) and SD1.3 (0.13 °C) (P = 0.053). This suggests that pigs housed at higher densities are less able to regulate their internal heat production. However, RR and performances were not significantly affected by heat load in this experimental set-up. A stocking density of 1.0 m2/animal may be sufficient to mitigate some negative effects of heat stress.

The impact of slaughter weight and sex on the carbon footprint of pig feed intake.

The impact of pork production on global livestock's greenhouse gas emissions is substantial. Understanding the factors influencing these emissions is crucial in achieving a more sustainable pig husbandry. In two independent experiments, the impact of slaughter weight on the carbon footprint (CFP) of pig feed intake (CFPFI) was evaluated for growing-finishing pigs of different sexes (entire males (EM), barrows (BA), immunocastrates (IC) and gilts (GI)). In experiment 1 118 animals were raised individually in experimental conditions. In experiment 2 384 animals were housed in group (four pigs per pen), in controlled commercial farm circumstances. All animals were fed ad libitum in a three-phase feeding regime and slaughtered at different BW, ranging from 110 to 148 kg (experiment 1) and from 99 to 138 kg (experiment 2). When only the fattening period was considered, the CFPFI was expressed per kg carcass growth. When the production of piglets was also taken into account, the CFPFI was calculated per kg carcass weight. For all sexes, the heavier the pig, the higher the CFPFI per kg carcass growth (P < 0.05, experiment 1; P < 0.001, experiment 2) and per kg carcass weight (P < 0.05, experiment 1; P < 0.01, experiment 2). In addition, BA had a significantly higher CFPFI per kg carcass growth and per kg carcass weight compared to EM and IC and to EM, IC and GI in respectively experiments 1 and 2 (P < 0.001). When performing a desk study with a soy-free (i.e. reduced CFP) feed in the third phase of experiment 2, slaughter weight no longer affected the CFPFI per kg carcass growth (P = 0.133) and per kg carcass weight (P = 0.856). A higher slaughter weight thus implies a lower sustainability, unless a sufficiently low-CFP feed is provided in the finisher phase.

The importance of pigs' castration strategy on carbon footprint of feed intake, nitrogen and phosphorus efficiency under different management conditions.

The environmental impact of livestock production is under scrutiny nowadays and is being situated both on a global level and local level. On a global level, the global warming potential (GWP) of meat production is criticised. On a local level, the excretion of nitrogen (N) and phosphorus (P) is of concern as excess nutrients may lead to water eutrophication and soil acidification. The ERA-NET project SuSI evaluated if immunocastration may act as an ecologically, socially and economic more sustainable alternative compared to pork production with surgical castrates (SCs) and boars (BOs). Here, we report the environmental impact of immunocastrates (ICs) vs SC and BO, based on four trials carried out by European SuSi partners. More specifically, we aimed to compare IC with SC and BO in terms of GWP, N and P excretion per kg of pork production and test if this differed between experiments. There was an interaction between trial and sex category (PTrial × Sex category < 0.005) for all environmental sustainability parameters. Surgical castrates performed worse (higher carbon footprint of the feed intake, N and P excretion, lower N and P efficiency) compared to IC and BO, but the size of the effect was trial dependent. Immunocastrates scored intermediate, with mostly no significant differences from BO in most trials, but with significantly better values compared to SC. Over trials, the carbon footprint of the feed intake (land use change inclusive) per kg lean meat gain in the growing-finishing phase was 9-16% lower for IC vs SC and 9-22% lower for BO vs SC. Nitrogen efficiency of IC and BO was 7-10% and 9-14%, respectively, higher compared to SC. Phosphorus efficiency of IC and BO was higher than that of SC by 6-14% and 9-17%, respectively. Per kg of lean meat gain in the growing-finishing phase, IC excreted between 14 and 19% less N and between 14 and 24% less P than SC. For BO, it was between 14 and 27% less N and between 14 and 31% less P than SC. Differences between trials were larger than differences between castration strategies and trial design may have amplified the observed effects. Improving feed efficiency, adapting the feed to the needs of the animal (avoiding excess nutrients) and choosing low-impact ingredients are key for improving the environmental sustainability of pig production. Ending physical castration is another step to attain this goal.

Consumer evaluation of meat quality from barrows, immunocastrates and boars in six countries.

The practice of surgical castration of piglets and its alternatives is still under debate. Production of boars may impair meat quality due to boar taint and reduced tenderness compared to meat from surgically castrated male pigs, while immunocastration reduces boar taint and may improve meat quality but seems to be less accepted by the pig chain. In this study, we aimed to evaluate the consumer's sensory appreciation of meat from barrows (BAs), immunocastrates (ICs) and boars (BOs) in six European countries, taking into account the selection of tainted carcass and consumers' appreciation of boar taint. Loin chops of 30 BAs, 30 ICs and 30 BOs were evaluated by 752 consumers in six countries: Belgium, Czech Republic, Poland, Portugal, Romania and Spain. Consumers rated odour, flavour, tenderness, juiciness, overall liking and willingness to buy and sensitivity to and liking of androstenone (AND) and liking of skatole (SKA) was also tested. In each of the six countries, consumers liked the odour of the BO samples less than that of BA, and IC intermediate. For flavour, tenderness, juiciness, overall liking and willingness to buy, liking scores given by the Czech, Polish and Portuguese consumers significantly differed between the BA, BO and IC. Willingness to buy was highest for BA by Czech and Polish consumers and for BA and IC by Portuguese consumers. The frequency of the negative check all terms that apply terms also differed, with a higher frequency of disgusting for BO compared to BA and IC and of off-flavour, irritating, manure, sweat, disappointing compared to BA, and intermediate for IC. 31% of the consumers disliked the odour of AND (NEGAND), and 36% of them were not sensitive; in contrast, 77% of the consumers disliked SKA (NEGSKA). The decrease in flavour liking score for BO compared to BA and IC was more outspoken by the NEGAND consumer, while NEGSKA consumers gave an overall lower liking score independent of the type of male pig. The results of this study indicate that IC can be a valid alternative for surgical castration.

Effect of dietary energy level in finishing phase on performance, carcass and meat quality in immunocastrates and barrows in comparison with gilts and entire male pigs.

Immunocastration, a technique consisting of two vaccinations against gonadotropin-releasing hormone (GnRH), can be used as alternative to surgical castration of piglets. It reduces boar taint and allows higher economic and ecological efficiency compared to barrows. The feeding strategy of immunocastrates, however, can still be improved. After second vaccination, when immunisation becomes fully effective, feed intake of immunocastrates increases sharply. This study aimed to investigate whether energy intake of immunocastrates after second vaccination could be reduced by lowering the dietary energy level of the finishing phase, without negatively affecting animal performance and quality of pork production. We hypothesised that immunocastrates already reach their limits in voluntary feed intake after second vaccination, and therefore would not be able to compensate the lower dietary energy level, in contrast to barrows. Therefore, this study aimed to assess the effect of high-energy diet (HE, net energy (NE) = 10.2 MJ/kg) compared to low-energy diet (LE, NE = 8.8 MJ/kg) in barrows and immunocastrates and as a reference, gilts and entire male pigs on a standard high-energy diet were included. CP and standardised ileal digestible amino acid levels were similar in both diets. For each treatment, eight pen replicates of six pigs per pen were evaluated on performance, carcass quality, meat and fat quality, digestibility, economic and ecological sustainability, behaviour and effectiveness of immune response. No difference in feed intake of immunocastrates between LE and HE could be demonstrated. As a result, daily energy intake of immunocastrates was higher on HE compared to LE, which resulted in a higher daily gain on HE. Feed conversion ratio (FCR) of immunocastrates on HE did not differ significantly with FCR of entire males. Barrows did not show higher average daily gain on HE compared to LE. Nitrogen efficiency was better in HE compared to LE, without negative effects on digestibility, carcass quality, economic parameters, behaviour or immune response. Small positive effects on the palatability of the meat of immunocastrates on HE were observed, although consumers did not prefer one of both feeds. Immunocastration was successful in reducing sexual and aggressive behaviour as well as in lowering the prevalence of boar taint from 15% in EM to 0% in immunocastrates. However, in two out of 96 immunocastrates (one on HE and one on LE), the immunocastration was not fully effective. In conclusion, this study did not show advantages of feeding immunocastrates or barrows a low-energy diet.

Colour-sidedness in Gloucester cattle is associated with a complex structural variant impacting regulatory elements downstream of KIT.

Colour-sidedness is a striking coat colour pattern found in a number of cattle breeds, typically characterised by a white stripe that extends along the back, head and belly of the animal. This dominant phenotype is caused by two related translocations (Cs6 and Cs29 ) that alter a region downstream of the KIT gene. Gloucester cattle are native to the UK and are known for an unusual colour-sided pattern that does not extend to the head. We carried out whole-genome sequencing of two Gloucester bulls as well as colour-sided Irish Moiled, British White and Pustertaler Sprinzen for comparison. We found that the Gloucester cattle also have a complex structural variant downstream of KIT, which overlaps the regions involved in Cs6 and Cs29 . All three alleles potentially disrupt a number of putative regulatory elements downstream of KIT. These results complement and expand on the recently published work focused on the Pinzgauer breed from Austria, a carrier of the same colour-sided pattern as seen in Gloucester cattle.

The effect of sex and slaughter weight on performance, carcass quality and gross margin, assessed on three commercial pig farms.

Economic margins on pig farms are small, and changing slaughter weights may increase farm profitability. However, one can question if the optimal slaughter weight is the same for each sex. On three farms, crossbred pigs (n = 1128) were used to determine the effect of sex and slaughter weight on performance, carcass quality and gross margin per pig place per year. On each farm, an equal number of entire males (EMs), barrows (BAs), immunocastrates (IC) and gilts (GIs) were housed separately in group pens. Pens were randomly divided into three categories of different slaughter weights: 105, 117 and 130 kg BW. In BA, the high average daily feed intake (ADFI) and the lower capacity to gain muscle led to a higher feed conversion ratio (FCR) and lower lean meat percentage in comparison to EM and IC. In all sexes, ADFI and FCR increased with an increasing slaughter weight but the effect of slaughter weight on carcass quality varied between sexes. In BA and GI, slaughter weight had no effect on carcass quality, but in EM and IC, carcass quality improved at higher slaughter weights. Gross margin per pig place per year was calculated as gross margin per pig × barn turnover per year, taking into account fixed costs per round, feed costs and output price per pig. The slaughter weight that gained the highest gross margin per year differed between sexes. Slaughtering BA and GI at 130 kg BW, compared to 105 or 117 kg BW, decreased the gross margin per pig place per year due to the lower margin per pig and barn turnover at higher weights. In IC and EM, no difference in gross margin per pig place per year could be demonstrated between slaughtering at 105, 117 or 130 kg BW. In IC, the increasing gross margin per pig with increasing slaughter weights counteracted with the lower barn turnover. In EM, gross margin per pig did not differ between slaughter weights, but the effect of barn turnover was too small to demonstrate significant differences between slaughter weights on gross margin per pig place per year. In conclusion, slaughter weight has an impact on profitability in BA and GI: they should not be slaughtered at 130 kg BW but at lower weights, but no effect could be demonstrated in EM and IC.

The effect of Piétrain sire on the performance of the progeny of two commercial dam breeds: a pig intervention study.

Genetic evaluation of Piétrain sires in Flanders occurs under standardized conditions, on test stations with fixed dam breeds, standardized diets and uniform management practices. As environmental conditions vary on commercial farms and differ from the test stations, this study aimed at understanding to what extent the sire, the dam breed and the interaction between both affects the translation of breeding values to practice. Dams of two commercial breeds were inseminated with semen from one of five different sires selected for contrasting breeding values (daily gain, feed conversion ratio and carcass quality). For each sire by dam breed combination, six pen replicates (with three gilts and three barrows per pen) were evaluated for growth performance from 9 weeks of age (20 kg) to slaughter (110 kg), and for carcass and meat quality. In our experimental setup, both sire and dam breed affected growth, carcass and meat quality traits. No significant sire×dam breed interactions on performance could be detected. Though a tendency for interaction on average daily feed intake between 20 and 110 kg (P=0.087), and on pork colour (lightness) (P=0.093) was present. In general, offspring of all tested sires behaved similarly in both dam breeds, indicating that estimated breeding values for Piétrain sires determined in one dam breed are representative in other dam breeds as well.

Review: Pork production with maximal nitrogen efficiency.

During growth, pigs convert plant protein into animal protein. The major part of the ingested protein is excreted via manure, with potential nitrogen (N) losses to the environment. To limit N losses and increase sustainability of pork production, the efficiency of protein conversion should be maximized. The aim of this paper is to critically evaluate diet and management strategies linked with N efficiency. Besides nutrition, we discuss three management strategies observed in science and in practice to be linked with improved N efficiency: genetic selection, castration and slaughter weight. Because diet has a marked effect on eventual N losses, it must also be taken into account when evaluating management strategies. A reductionist approach, such as feeding the same diet across all management treatments, may overestimate the effect of a management strategy and eventually lead to incorrect conclusions. The amount of excreted N depends on the amount of ingested N, the amount of absorbed N, the amino acid (AA) balance in the diet and the animal's N and AA requirements. Daily multiphase feeding adapted to the individual animal's AA needs is likely to be the most N efficient. For animals housed in groups, phase feeding is necessary. When combined with periods of temporary AA restriction, N efficiency can be further improved. Specific AA consumption must be balanced by applying the ideal protein concept. With better knowledge of the requirements of individual animals and the commercial availability of certain AAs, the total dietary CP level can be lowered within limits. Further research is needed on the minimal CP level that allows maximal performance. For this end a useful parameter may be the ratio of standardized ileal digestible (SID) lysine : apparent total tract digestible CP level. By combining optimal nutrition and management, a whole body N efficiency approaching 60% may be achievable in the near future.

The effect of GnRH vaccination on performance, carcass, and meat quality and hormonal regulation in boars, barrows, and gilts.

The aim of this study was to assess the effect of GnRH vaccination in boars (BO), barrows (BA), and gilts (GI) slaughtered at an average BW of 133 kg. Forty BO, 40 BA, and 40 GI were housed individually, fed ad libitum, and divided into 2 groups of 20 pigs per sex, a control group (CON) and a GnRH vaccinated group (IC). The IC group received 2 injections of GnRH vaccine (Improvac) at 70 and 105 kg. In BA, GnRH vaccination had minor effects on performance. Observed effects in BO and GI are therefore probably not caused by the GnRH vaccination itself but by the subsequent withdrawal of gonadal hormones. In BO, the increased feed intake after GnRH vaccination ( < 0.001), accompanied by a fall in estradiol and testosterone levels, led to faster growth ( = 0.014), increased back fat thickness ( = 0.021), and a tendency for a decreased meat percentage ( = 0.052). GnRH vaccination of BO decreased the boar taint-related sensory attribute scores similar to the levels of BA and GI ( < 0.001). In GI, the increased feed intake after GnRH vaccination ( < 0.001), accompanied by low progesterone levels, led to faster growth ( < 0.001), increased back fat thickness ( = 0.018), decreased meat percentage ( = 0.032), and a decreased shear force ( = 0.002) without significant differences in the sensory profile.

Immunocastrated male pigs: effect of 4 v. 6 weeks time post second injection on performance, carcass quality and meat quality.

Immunocastration or vaccination against boar taint can be used as alternative for surgical castration of male piglets. The vaccine is administrated twice. After the second vaccination (V2), the pigs behave like barrows instead of boars and their feed intake increases which may result in a lower lean meat percentage. The timing of V2 is therefore crucial to find the right balance between the advantages of entire males and barrows. In this study, we evaluated the effect of time post second injection within the advised time frame (4 v. 6 weeks before slaughter) on behaviour, performance, carcass and meat quality of immunocastrated male pigs. In total, 180 animals (hybrid sow×Piétrain): 60 gilts, 60 male pigs vaccinated 6 weeks before slaughter (IM-6) and 60 male pigs vaccinated 4 weeks before slaughter (IM-4), all slaughtered at comparable slaughter weights. After 20 weeks of age, IM-6 showed more inactive behaviour at the expense of playing and aggressive behaviour. Daily feed intake (DFI), daily gain (DG) and feed conversion ratio (FCR) did not differ significantly between IM-6 and IM-4. Gilts had a lower DFI and DG in the late finishing phase and a higher FCR overall compared with both IM groups. Gilts showed a higher lean meat content compared with both IM groups. Earlier vaccination increased dressing percentage, which could partly be explained by the lower weight of the gastrointestinal tract, but not by testes weight. Meat quality traits and palatability did not differ significantly between IM-6 and IM-4. Vaccination of immunocastrates at 6 compared with 4 weeks before slaughter improved the calmness in the stable and the dressing percentage, while maintaining performance and carcass characteristics.

The effect of the MC4R gene on boar taint compounds, sexual maturity and behaviour in growing-finishing boars and gilts.

Societal pressure to ban surgical castration of male piglets is rising due to animal welfare concerns, thus other methods to prevent boar taint need to be explored. Genetic selection against boar taint appears to be a long-term sustainable alternative. However, as boar taint is linked to reproductive hormones, it is important to consider possible negative side effects such as delayed sexual maturity or changes in behaviour. We reported earlier that the melanocortin-4 receptor (MC4R) marker can be used to reduce boar taint levels in fat of boars. The objective of this study was to evaluate whether MC4R marker-assisted selection for lower boar taint prevalence affects plasma levels of boar taint compounds and testosterone; sexual maturity; behaviour; skin lesions; and lameness in boars and gilts. Using an intervention study with a 2×2 design, 264 boars and gilts differing on position 893 of the MC4R gene (AA v. GG) were compared. The MC4R polymorphism did not affect the plasma concentration of either androstenone or testosterone at different time points, whereas the concentration of skatole was significantly lower (P=0.003) and the concentration of indole tended to be lower (P=0.074) in GG compared with AA boars. A higher percentage of gilts of the GG genotype were in puberty at slaughter age compared with AA gilts (P<0.001). The age of the boars at sexual maturity (as indicated by the first positive preputial smear test) did not differ between AA and GG boars. In contrast, weight of GG boars at sexual maturity tended to be lower (P=0.065). During the period from 6 weeks of age to slaughter, boars and gilts of the GG genotype showed more playing behaviour (P=0.015) and less passive and feeding behaviour (P=0.003). They showed more skin lesions on their back and caudal area (P=0.022), and tended to show more skin lesions on their head and anterior area (P=0.093) compared with AA animals. In conclusion, the polymorphism in the MC4R gene can be used as a marker without negative effects on reproduction characteristics in boars and gilts. Genetic selection towards a lower prevalence of boar taint will lead to more active pigs with more skin lesions. Management strategies may therefore be necessary to reduce skin lesions in the selected animals.

An intervention study demonstrates effects of MC4R genotype on boar taint and performances of growing-finishing pigs.

The Asp298Asn polymorphism of the melanocortin-4 receptor (MC4R) in pigs is known to affect economically important traits such as growth rate and backfat thickness. We have assessed the possible use of this polymorphism as a molecular marker to perform genetic selection toward lower boar taint levels without compromising growth performance and carcass and meat quality in commercial boars and gilts. Homozygous boars and gilts of the AA genotype and GG genotype were compared in an intervention study with a 2 × 2 design to assess main effects and possible interactions between sex and genotype. The concentrations of the 3 boar taint compounds androstenone ( = 0.044), skatole ( = 0.049), and indole ( = 0.006) were significantly higher in fat of AA boars compared to GG boars. However, no effect on the sensory analysis of the fat samples could be observed. Between 20 and 115 kg BW, AA pigs showed higher ADFI than GG pigs ( < 0.001). An interaction between genotype and sex was observed for ADG ( = 0.044): AA boars had a significantly higher ADG than GG boars but there was no significant difference between the gilts. Daily lean meat gain tended to be higher in boars compared to gilts ( = 0.051), independent of genotype. Similarly, boars showed higher G:F compared to gilts ( < 0.001), without effect of genotype. Genotype and sex affected several carcass quality parameters but there was no interaction. Pigs of the AA genotype displayed a lower dressing percentage ( = 0.005), lower ham width ( = 0.024), lower muscle thickness ( = 0.011), and higher fat thickness ( < 0.001), resulting in a lower lean meat percentage ( < 0.001) in comparison with GG pigs. Gilts had a significantly higher dressing percentage ( < 0.001), higher muscle thickness ( < 0.001), higher ham width ( < 0.001), and lower ham angle ( < 0.001) compared to boars. Other than the boar taint compounds, meat quality was not affected by genotype. Pork of gilts was darker ( = 0.014) and less exudative during cooking ( < 0.001) and contained more intramuscular fat ( = 0.013). These results indicate that genetic selection against boar taint is possible using this marker. This will also result in lower feed intake and ADG and, consequently, better carcass quality.

Development of a quantitative method for the simultaneous analysis of the boar taint compounds androstenone, skatole and indole in porcine serum and plasma by means of ultra-high performance liquid chromatography coupled to high resolution mass spectrometry.

Boar taint is an off-odour occurring while heating meat or fat from boars. A method detecting the three compounds (androstenone, skatole and indole) simultaneously in blood would offer substantial advantages since it would allow monitoring the impact of rearing strategies. Therefore, a UHPLC-HR-Orbitrap-MS analysis method is optimized and validated for the quantification of these compounds in plasma or serum. Sample pre-treatment involved an extraction with diethylether followed by a centrifugal filtration (30 kDa). Limits of detection and quantification varied between 0.5 and 1 µg L(-1) and 2 and 3 µg L(-1) for the three compounds, respectively. Besides, an excellent repeatability (RSD < 7.6%), within-laboratory reproducibility (RSD<10.5%), recovery (87-97%) and linearity (R(2)>0.99) were recorded. Correlations between serum/plasma and fat levels of the boar taint compounds were positive for skatole (r(serum) = 0.39 and r(plasma) = 0.84) and androstenone (r(serum) = 0.73-0.78 and r(plasma) = 0.32-0.80).

Prolonged exposure to IL-1beta and IFNgamma induces necrosis of L929 tumor cells via a p38MAPK/NF-kappaB/NO-dependent mechanism.

Interleukin-1beta (IL-1beta) is a cytokine that shares with tumor necrosis factor (TNF) the ability to initiate largely similar signaling pathways, leading to proinflammatory gene expression. In contrast to TNF, however, IL-1beta is not believed to induce tumor cell death. Here we demonstrate that prolonged treatment with IL-1beta, in combination with interferon-gamma (IFNgamma), is cytotoxic for L929 tumor cells. IL-1beta/IFNgamma-induced cytotoxicity requires only minimal amounts of IL-1beta and shows morphological features of necrosis. Although TNF induces a similar response, we could exclude a contribution of endogenous TNF production in the effect of IL-1beta/IFNgamma. Cell death in response to IL-1beta/IFNgamma is independent of caspases, but requires the IL-1beta/IFNgamma-induced production of inducible nitric oxide synthase (iNOS) and NO. Moreover, necrosis and iNOS/NO production could be prevented by treatment of the cells with a p38 mitogen activated protein kinase (p38MAPK) or IkappaB kinase beta inhibitor. Altogether, these findings demonstrate that prolonged exposure to IL-1beta plus IFNgamma induces L929 tumor cell necrosis, via a p38MAPK and nuclear factor-kappaB (NF-kappaB)-dependent signaling pathway, leading to the expression of iNOS and the production of toxic NO levels.

CD154 costimulated ovine primary B cells, a cell culture system that supports productive infection by bovine leukemia virus.

Bovine leukemia virus (BLV) is closely associated with the development of B-cell leukemia and lymphoma in cattle. BLV infection has also been studied extensively in an in vivo ovine model that provides a unique system for studying B-cell leukemogenesis. There is no evidence that BLV can directly infect ovine B cells in vitro, and there are no direct data regarding the oncogenic potential of the viral Tax transactivator in B cells. Therefore, we developed ovine B-cell culture systems to study the interaction between BLV and its natural target, the B cell. In this study, we used murine CD154 (CD40 ligand) and gamma-chain-common cytokines to support the growth of B cells isolated from ovine lymphoid tissues. Integrated provirus, extrachromosomal forms, and viral transcripts were detected in BLV-exposed populations of immature, rapidly dividing surface immunoglobulin M-positive B cells from sheep ileal Peyer's patches and also in activated mature B cells isolated from blood. Conclusive evidence of direct B-cell infection by BLV was obtained through the use of cloned B cells derived from sheep jejunal Peyer's patches. Finally, inoculation of sheep with BLV-infected cultures proved that infectious virus was shed from in vitro-infected B cells. Collectively, these data confirm that a variety of ovine B-cell populations can support productive infection by BLV. The development of ovine B-cell cultures permissive for BLV infection provides a controlled system for investigating B-cell leukemogenic processes and the pathogenesis of BLV infection.

Tumor-selective gene transduction and cell killing with an oncotropic autonomous parvovirus-based vector.

A recombinant MVMp of the fibrotropic strain of minute virus of mice (MVMp) expressing the chloramphenicol acetyltransferase reporter gene was used to infect a series of biologically relevant cultured cells, normal or tumor-derived, including normal melanocytes versus melanoma cells, normal mammary epithelial cells versus breast adenocarcinoma cells, and normal neurons or astrocytes versus glioma cells. As a reference cell system we used normal human fibroblasts versus the SV40-transformed fibroblast cell line NB324K. After infection, we observed good expression of the reporter gene in the different tumor cell types, but only poor expression if any in the corresponding normal cells. We also constructed a recombinant MVMp expressing the green fluorescent protein reporter gene and assessed by flow cytometry the efficiency of gene transduction into the different target cells. At a multiplicity of infection of 30, we observed substantial transduction of the gene into most of the tumor cell types tested, but only marginal transduction into normal cells under the same experimental conditions. Finally, we demonstrated that a recombinant MVMp expressing the herpes simplex virus thymidine kinase gene can, in vitro, cause efficient killing of most tumor cell types in the presence of ganciclovir, whilst affecting normal proliferating cells only marginally if at all. However, in the same experimental condition, breast tumor cells appeared to be resistant to GCV-mediated cytotoxicity, possibly because these cells are not susceptible to the bystander effect. Our data suggest that MVMp-based vectors could prove useful as selective vehicles for anticancer gene therapy, particularly for in vivo delivery of cytotoxic effector genes into tumor cells.

CD40 signaling induces B cell responsiveness to multiple members of the gamma chain-common cytokine family.

CD40 signaling induces B cell proliferative and differentiation responses that can be modulated by many different cytokines. Cytokines in the IL-2 receptor gamma chain (gammac)-common family are known to play an integral role in B cell development. Therefore, we investigated the possibility that CD40 signaling induced B cell responsiveness to multiple gammac-common cytokines and that individual gammac-common cytokines induced distinct B cell responses. B cells were isolated from lymphoid follicles of sheep Peyer's patches (PP) and co-cultured with murine CD40 ligand (mCD40L). CD40 signaling induced PP B cell responsiveness to recombinant human IL-2, IL-4, IL-7 and IL-15. mCD40L-induced B cell growth was enhanced by combining IL-4 with a second gammac-common cytokine and sustained B cell growth required co-stimulation with IL-4 plus IL-2, IL-7 and IL-15. gammac-common cytokine responsiveness remained dependent upon CD40 signaling, and removal of mCD40L resulted in B cell differentiation and cell death. Similar proliferative responses to mCD40L and gammac-common cytokines were observed for both immature (ileal) and mature (jejunal) PP B cells. Finally, the capacity of CD40-activated B cells to respond to multiple gammac-common cytokines was analyzed with individual PP B cell clones. All B cell clones displayed similar proliferative responses to IL-2 but quantitatively different responses to IL-4, IL-7 and IL-15. The biological significance of B cell responsiveness to multiple gammac-common cytokines is discussed.

Human immunodeficiency virus type 2 produces a defect in CD3-gamma gene transcripts similar to that observed for human immunodeficiency virus type 1.

T cells are central players in the immune response to infectious disease, with the specificity of their responses controlled by the T-cell receptor (TCR)/CD3 complex on the cell surface. Impairment of TCR/CD3-directed CD4(+) T-cell immune responses is frequently observed in individuals infected with human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). Virus replication is also regulated by T-cell activation factors, with HIV-1 and HIV-2 responding to different TCR/CD3-directed cellular pathways. We previously demonstrated that HIV-1 infection of the human interleukin-2-dependent CD4(+) T-cell line WE17/10 abrogates TCR/CD3 function and surface expression by a specific loss of CD3-gamma gene transcripts. In this study, we show that HIV-2 provokes the same molecular defect in CD3-gamma gene transcripts, resulting in a similar but delayed progressive loss of TCR/CD3 surface expression after infection.