The effects of stunning methods on product qualities in force-fed ducks and geese. 1. Carcass downgrading and meat quality

This study investigated the effects of various stunning methods on the quality of carcass and meat in ducks and geese force-fed for the production of French 'foie gras'. The ducks (n = 30 per group) were stunned with one of the following techniques: electrical stunning in a water bath (50 Hz AC, 130 mA, 4 s), head-only electrical stunning (50 Hz AC, 600 mA, 4 s), mechanical stunning (captive bolt) and controlled atmosphere stunning (CAS: phase 1, CO2 (40%)-O2 (30%)-N2 (30%), for 2 min followed by phase 2, CO2 > 85% in air, for 2 min). The same methods (except head-only stunning which was not applied) were used in geese (n = 40 per group). During the first 5 min after slaughter, the stunning techniques that did not kill the animals were associated with a high incidence of head movements (mechanical and electrical head-only stunning), convulsions and convulsive wing flapping (mechanical stunning), in both species. Consequently, the rate of post-mortem pH fall in breast muscle was enhanced and the meat was paler when measured at 24 h or 6 days post mortem (L*, a*, b* coordinates). In ducks, the animals stunned in the water bath showed the lowest bleeding efficiency, compared to the three other procedures. In geese, the mechanical stunning allowed the highest recovery of blood compared to the CAS and the water-bath methods. Meat texture assessed instrumentally and fluid losses during storage and processing were not affected by the stunning method in any species. Sensory analysis showed a higher score for bloody appearance of raw meat in ducks stunned with the methods that kill before neck cutting (water-bath and CAS). This effect was, however, not linked to the rate of bleeding. The sensory properties of cooked meat were not affected by the stunning method. In both ducks and geese, CAS was associated with the lowest rate of fractures of humeral bone but CAS-stunned geese showed the most engorged wing veins. Overall, these results show the positive effect of CAS on the appearance defects of carcass and meat of ducks and geese, and, on the contrary, they confirm the detrimental effects of water-bath stunning on these criteria. However, the incidences on meat sensory qualities were scarce.     

Anti-human immunodeficiency virus type 1 humoral immune response and highly active antiretroviral treatment

Highly active antiretroviral treatment (HAART) of human immunodeficiency type 1 (HIV-1) infection is very effective in controlling infection, but elimination of viral infection has not been achieved as yet, and upon treatment interruption an immediate rebound of viremia is observed. A combination of HAART with an immune stimulation might allow treatment interruption without this rebounding viremia, as the very low viremias observed with successful HAART may be insufficient to permit maintenance of a specific anti-HIV-1 immune response. The objective of this study was to compare the humoral immune response of individuals undergoing successful HAART (NF=no failure) with that of individuals with evidence of failure of therapy (FT) and to verify if the viremia peaks observed in individuals with therapy failure would act as a specific stimulus for the humoral anti-HIV-1 immune response. Antibodies binding to gp120 V3 genotype consensus peptides were more frequently observed for FT, mainly against peptides corresponding to sequences of genotypes prevalent in the Rio de Janeiro city area, B and F. HIV-1 neutralization of HIV-1 IIIB and of four primary isolates from Rio de Janeiro was less frequently observed for plasma from the NF than the FT group, but this difference was more expressive when plasma from individuals with detectable viremia were compared to that of individuals with undetectable viral loads in the year before sample collection. Although statistically significant differences were observed only in some specific comparisons, the study indicates that presence of detectable viremia may contribute to the maintenance of a specific anti-HIV-1 humoral immune response.     

Antioxidant activity of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> cells grown in continuous culture as a function of their carotenoid and fatty acid content

The influence of culture conditions on the quality of Haematococcus pluvialis biomass is assessed. Continuously grown cells have been characterised with respect to their astaxanthin, fatty acid content, and antioxidant activity and compared with those of non-growing haematocysts. Moderate limitation of nitrate availability (1.7 mM) under continuous growth conditions favoured the production of reddish palmelloid cells whose extracts possessed antioxidant activity equivalent to that of haematocyst extracts, despite the lower astaxanthin content (0.6%d.wt.), which is compensated by a higher fatty acid level (7.6%d.wt.). Green cells produced under nitrate saturation conditions (>4.7 mM) exhibit only 40% antioxidant activity than palmelloid. In addition, the major fatty acid present in palmelloid cells was oleic acid (40%f.a.), whereas, in both green cells and haematocysts, the main fatty acids were myristic, palmitic, and oleic acid (20–30%f.a. each). Biomass extracts were fractionated and analysed. The antioxidant capacity was a function of both the carotenoid and the fatty acid profiles, the antioxidant capacity of astaxanthin diesters fraction being 60% higher than astaxanthin monoesters fraction and twice than free astaxanthin. In such a way, the evaluation of the quality of H. pluvialis biomass must take into account both variables. When considering the production of H. pluvialis biomass for human consumption, special attention should be paid to the one-step continuous system approach for the generation of cells rich in both astaxanthin and fatty acids, as they have high antioxidant activity but without thick hard cell wall.     

Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression

The majority of people infected with hepatitis C virus (HCV) fail to generate or maintain a T-cell response effective for viral clearance. Evidence from murine chronic viral infections shows that expression of the coinhibitory molecule PD-1 predicts CD8+ antiviral T-cell exhaustion and may contribute to inadequate pathogen control. To investigate whether human CD8+ T cells express PD-1 and demonstrate a dysfunctional phenotype during chronic HCV infection, peripheral and intrahepatic HCV-specific CD8+ T cells were examined. We found that in chronic HCV infection, peripheral HCV-specific T cells express high levels of PD-1 and that blockade of the PD-1/PD-L1 interaction led to an enhanced proliferative capacity. Importantly, intrahepatic HCV-specific T cells, in contrast to those in the periphery, express not only high levels of PD-1 but also decreased interleukin-7 receptor alpha (CD127), an exhausted phenotype that was HCV antigen specific and compartmentalized to the liver, the site of viral replication.     

Characterization of heme as activator of Toll-like receptor 4

Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an atom of iron linked to four ligand groups of porphyrin. A high amount of free heme, a potential amplifier of the inflammatory response, is a characteristic feature of diseases with increased hemolysis or extensive cell damage. Here we demonstrate that heme, but not its analogs/precursors, induced tumor necrosis factor-alpha (TNF-alpha) secretion by macrophages dependently on MyD88, TLR4, and CD14. The activation of TLR4 by heme is exquisitely strict, requiring its coordinated iron and the vinyl groups of the porphyrin ring. Signaling of heme through TLR4 depended on an interaction distinct from the one established between TLR4 and lipopolysaccharide (LPS) since anti-TLR4/MD2 antibody or a lipid A antagonist inhibited LPS-induced TNF-alpha secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF-alpha and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferon-inducible protein-10 secretion or co-stimulatory molecule expression. These findings support the concept that the broad ligand specificity of TLR4 and the different activation profiles might in part reside in its ability to recognize different ligands in different binding sites. Finally, heme induced oxidative burst, neutrophil recruitment, and heme oxygenase-1 expression independently of TLR4. Thus, our results presented here reveal a previous unrecognized role of heme as an extracellular signaling molecule that affects the innate immune response through a receptor-mediated mechanism.     

CD101 surface expression discriminates potency among murine FoxP3+ regulatory T cells

CD4+CD25+FoxP3+ regulatory T cells (Treg) have been shown to be protective in animal models of autoimmunity and acute graft-vs-host disease. However, owing to the functional heterogeneity among CD4+CD25+ T cells, surface markers expressed selectively on functionally active Treg would be useful for purposes of identifying and isolating such cells. We generated a rabbit mAb against murine CD101, a transmembrane glycoprotein involved in T cell activation. Among freshly isolated T cells, CD101 was detected on 25-30% of CD4+CD25+ Treg and approximately 20% of conventional memory T cells. CD101(high) Treg displayed greater in vitro suppression of alloantigen-driven T cell proliferation as compared with CD101(low) Treg. In a model of graft-vs-host disease induced by allogeneic bone marrow transplantation in vivo bioluminescence imaging demonstrated reduced expansion of donor-derived luciferase-labeled conventional T cells in mice treated with CD101(high) Treg, compared with CD101(low) Treg. Moreover, treatment with CD101(high) Treg resulted in improved survival, reduced proinflammatory cytokine levels and reduced end organ damage. Among the CD101(high) Treg all of the in vivo suppressor activity was contained within the CD62L(high) subpopulation. We conclude that CD101 expression distinguishes murine Treg with potent suppressor activity.     

N-linked glycosylation status of classical swine fever virus strain Brescia E2 glycoprotein influences virulence in swine

E2 is one of the three envelope glycoproteins of classical swine fever virus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 were modified by site-directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classical swine fever live attenuated vaccines.     

A polyvalent vaccine for high-risk prostate patients: “are more antigens better?”

We have shown the immunogenicity and safety of synthetic carbohydrate vaccines when conjugated to the carrier keyhole limpet hemocyanin (KLH) and given with the adjuvant, QS-21, in patients with biochemically relapsed prostate cancer. To determine whether immune response could be further enhanced with stimulation by multiple antigens, a hexavalent vaccine was prepared using previously determined doses and administered in a Phase II setting to 30 high-risk patients. The hexavalent vaccine included GM2, Globo H, Lewis^y, glycosylated MUC-1-32mer and Tn and TF in a clustered formation, conjugated to KLH and mixed with QS-21. Eight vaccinations were administered over 13 months. All 30 patients had significant elevations in antibody titers to at least two of the six antigens; 22 patients had increased reactivity with FACS. These serologic responses were lower than that seen previously in patients treated with the respective monovalent vaccines. The reciprocal median combined IgM and IgG antibody titers with ELISA against MUC1, Tn, TF, globo H and GM2 for these 30 patients were 640, 80, 120, 40 and 0, compared to 1280, 640, 1280, 320 and 160 seen in patients receiving individual monovalent vaccines. This hexavalent vaccine of synthetic “self” antigens broke immunologic tolerance against two or more antigens in all 30 vaccinated patients, was safe, but antibody titers against several of the antigens were lower than those seen in individual monovalent trials. No impact on PSA slope was detected. We address the relevance of the multivalent approach for prostate cancer treatment. Supported by the Prostate Cancer Foundation, The PepsiCo Foundation, The Sharon Hels and Brad Reed Fund, Swim Across America, The Sara Chait Foundation. Dr. Philip Livingston is a consultant for and shareholder in Progenics Pharmaceuticals, Inc.     

Nitric oxide, mitochondrial hyperpolarization, and T cell activation

T lymphocyte activation is associated with nitric oxide (NO) production, which plays an essential role in multiple T cell functions. NO acts as a messenger, activating soluble guanyl cyclase and participating in the transduction signaling pathways involving cyclic GMP. NO modulates mitochondrial events that are involved in apoptosis and regulates mitochondrial membrane potential and mitochondrial biogenesis in many cell types, including lymphocytes. Mitochondrial hyperpolarization (MHP), an early and reversible event during both activation and apoptosis of Tlymphocytes, is regulated by NO. Here, we discuss recent evidence that NO-induced MHP represents a molecular switch in multiple T cell signaling pathways. Overproduction of NO in systemic lupus erythematosus induces mitochondrial biogenesis and alters Ca(2+) signaling. Thus, whereas NO plays a physiological role in lymphocyte cell signaling, its overproduction may disturb normal T cell function, contributing to the pathogenesis of autoimmunity.