Biological roles of host defense peptides: lessons from transgenic animals and bioengineered tissues.

Host defense peptides (HDPs) have long been recognized as microbicidal agents, but their roles as modulators of innate and adaptive immunity have only more recently been appreciated. The study of transgenic animal and tissue models has provided platforms to improve our understanding of the immune modulatory functions of HDPs. Here, the characterization of transgenic animals or tissue models that over-express and/or are deficient for specific HDPs is reviewed. We also attempt to reconcile this data with evidence from human studies monitoring HDP expression at constitutive levels and/or in conjunction with inflammation, infection models, or disease states. We have excluded activities ascribed to HDPs derived exclusively from in vitro experiments. An appreciation of the way that HDPs promote innate immunity or influence the adaptive immune response is necessary in order to exploit their therapeutic or adjuvant potential and to open new perspectives in understanding the basis of immunity. The potential applications for HDPs are discussed.

A comparison between isolated blood dendritic cells and monocyte-derived dendritic cells in pigs.

Various dendritic cell (DC) populations exist that differ in phenotype and ability to present antigen to T cells. For example, plasmacytoid DCs (pDCs) are less potent T cell activators compared with conventional DCs (cDCs). Here, we compared porcine blood DCs (BDCs), containing pDCs and cDCs, and monocyte-derived DCs (MoDC), consisting of cDCs, in their phenotype, ability to uptake antigen, activation and maturation and their ability to present antigen to autologous T cells. Pigs represent an important animal model, whose immune system in many respects closely resembles that of humans. For example, the distribution of Toll-like receptors is similar to that of humans, in contrast to that of mice. Here we demonstrate that both populations endocytose foreign material. Following lipopolysaccharide stimulation, CD80/86 and chemokine receptor (CCR)7 expression was increased in both populations as was the expression of the chemokine ligands (CCL)-2, CCL-4, CCL-20 and CXCL-2. Although basal and post-stimulation protein concentrations of interleukins 6 and 8 and tumour necrosis factor-alpha were higher in MoDCs, protein concentrations showed a higher fold increase in BDCs. Antigen-specific proliferation of autologous T cells was induced by MoDCs and BDCs. Interestingly, while MoDCs induced stronger proliferation in naive T cells, no difference in proliferation was observed when primed T cells were studied. These results demonstrate that isolated porcine BDCs are highly responsive to stimulation with lipopolysaccharide and are functionally able to drive primed T-cell proliferation to the same extent as MoDCs.

Strategies to link innate and adaptive immunity when designing vaccine adjuvants.

Adjuvants are important components of vaccine formulations. Their functions include the delivery of antigen, recruitment of specific immune cells to the site of immunization, activation of these cells to create an inflammatory microenvironment, and maturation of antigen-presenting cells for enhancement of antigen-uptake and -presentation in secondary lymphoid tissues. Adjuvants include a large family of molecules and substances, many of which were developed empirically and without knowledge of their specific mechanisms of action. The discovery of pattern recognition receptors including Toll-like-, nucleotide-binding oligomerization domain (NOD)- and mannose-receptors, has significantly advanced the field of adjuvant research. It is now clear that effective adjuvants link innate and adaptive immunity by signaling through a combination of pathogen recognition receptors (PRRs). Research in our lab is focused towards the development of novel adjuvants and immunomodulators that can be used to improve neonatal vaccines for humans and animals. Using a neonatal pig model for pertussis, we are currently analyzing the effectiveness of host defence peptides (HDPs), bacterial DNA and polyphosphazenes as vaccine adjuvants.

The effects of conjugated linoleic acid supplementation during resistance training.

PURPOSE: We determined the effects of conjugated linoleic acid (CLA) supplementation during resistance training. METHODS: Seventy-six subjects were randomized to receive CLA (5 g.d(-1)) or placebo (PLA) for 7 wk while resistance training 3 d.wk(-1). Seventeen subjects crossed over to the opposite group for an additional 7 wk. Measurements at baseline, 7 wk, and 14 wk (for subjects in the crossover study) included body composition, muscle thickness of the elbow flexors and knee extensors, resting metabolic rate (RMR), bench and leg press strength, knee extension torque, and urinary markers of myofibrillar degradation (3-methylhistidine (3MH) and bone resorption (cross-linked N-telopeptides (Ntx)). RESULTS: After 7 wk the CLA group had greater increases in lean tissue mass (LTM) (+1.4 vs +0.2 kg; P < 0.05), greater losses of fat mass (-0.8 vs +0.4 kg; P < 0.05), and a smaller increase in 3MH (-0.1 vs + 1.3 micromol.kg LTM.d(-1); P < 0.05) compared with PLA. Changes between groups were similar for all other measurements, except for a greater increase in bench press strength for males on CLA (P < 0.05). In the crossover study subjects had minimal changes in body composition, but smaller increases in 3MH (-1.2 vs +2.2 micromol.kg LTM.d(-1); P < 0.01) and NTx (-4.8 vs +7.3 nmol.kg(-1) LTM.d(-1); P < 0.01) while on CLA versus PLA. CONCLUSIONS: Supplementation with CLA during resistance training results in relatively small changes in body composition accompanied by a lessening of the catabolic effect of training on muscle protein.

Porcine neonatal blood dendritic cells, but not monocytes, are more responsive to TLRs stimulation than their adult counterparts.

The neonatal immune system is often considered as immature or impaired compared to the adult immune system. This higher susceptibility to infections is partly due to the skewing of the neonatal immune response towards a Th2 response. Activation and maturation of dendritic cells (DCs) play an important role in shaping the immune response, therefore, DCs are a target of choice for the development of efficient and protective vaccine formulations able to redirect the neonatal immune response to a protective Th1 response. As pigs are becoming more important for vaccine development studies due to their similarity to the human immune system, we decided to compare the activation and maturation of a subpopulation of porcine DCs in adult and neonatal pigs following stimulation with different TLR ligands, which are promising candidates for adjuvants in vaccine formulations. Porcine blood derived DCs (BDCs) were directly isolated from blood and consisted of a mix of conventional and plasmacytoid DCs. Following CpG ODN (TLR9 ligand) and imiquimod (TLR7 ligand) stimulation, neonatal BDCs showed higher levels of expression of costimulatory molecules and similar (CpG ODN) or higher (imiquimod) levels of IL-12 compared to adult BDCs. Another interesting feature was that only neonatal BDCs produced IFN-α after TLR7 or TLR9 ligand stimulation. Stimulation with CpG ODN and imiquimod also induced enhanced expression of several chemokines. Moreover, in a mixed leukocyte reaction assay, neonatal BDCs displayed a greater ability to induce lymphoproliferation. These findings suggest that when stimulated via TLR7 or TLR9 porcine DCs display similar if not better response than adult porcine DCs.

Stability of expression of reference genes in porcine peripheral blood mononuclear and dendritic cells.

Real-time quantitative PCR (RT-qPCR) is a critical tool used to evaluate changes in gene expression. The precision of this tool is reliant upon the selection of reference genes whose expression remains unaltered in culture conditions and following stimulation. Stably expressed reference genes are used to normalize data so observed changes in expression are not due to artifacts but rather reflect physiological changes. In this study, we examined the expression stability of the porcine genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), eukaryotic elongation factor 1 gamma-like protein (eEF1), ribosomal protein L19 (RPL19), beta-actin (ACTB) and ATP synthase mitochondrial F0 complex (ATP5G1) in peripheral blood mononuclear cells (PBMCs), monocytes, monocyte-derived dendritic cells (MoDCs), blood isolated dendritic cells (BDCs) and T cells with or without stimulation with lipolysaccharide (LPS). An M value was used as a measure of gene stability as determined using geNORM software. Recommendations for the use of reference genes include using GAPDH and B-actin in PBMCs: RPL19 and SDHA in T cells; RPL19 and B-actin in monocytes; RPL-19 and SDHA in BDCs: and RPL-19 and ATP5GA in MoDCs.

Differential activation and maturation of two porcine DC populations following TLR ligand stimulation.

Dendritic cells (DCs) are at the interface of innate and adaptive immune responses. Once activated via triggering of their pattern recognition receptors (PRRs), they acquire a mature state and migrate to the lymph nodes where they activate T cells and direct the immune response. Compounds that trigger PRRs are potential vaccine adjuvants, hence in this study we stimulated two porcine DC populations, namely monocyte-derived DCs (MoDCs) and blood DCs (BDCs), with a broad range of toll-like receptors (TLRs) ligands and assessed the activation/maturation state of these porcine DCs. In order to determine if TLR ligands would have an effect on porcine DCs, we characterized the expression of TLRs and demonstrated that MoDCs and BDCs expressed the same set of TLRs but at different levels. Of the TLR ligands examined, lipopolysaccharide (LPS) and poly I:C were the most potent activators of MoDCs, inducing the up-regulation of co-stimulatory molecules CD80/86 and the chemokine receptor CCR7, and production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)alpha. The most effective in inducing BDCs activation were LPS and class A CpG oligodeoxynucleotide (ODN), resulting in up-regulation of chemokine receptor (CCR)7 and down-regulation of CCR2 and CCR5, production of IL-12p40, and expression of a broad range of chemokines that were able to attract porcine immune cells.

Protein supplementation before and after resistance training in older men.

We determined the effects of protein supplementation immediately before (PRO-B) and after (PRO-A) resistance training (RT; 12 weeks) in older men (59-76 years), and whether this reduces deficits in muscle mass and strength compared to younger men (18-40 years). Older men were randomized to PRO-B (0.3 g/kg protein before RT + placebo after RT, n=9), PRO-A (placebo before + protein after RT, n=10), or PLA (placebo before and after RT, n=10). Lean tissue mass, muscle thickness of the elbow, knee, and ankle flexors and extensors, and leg and bench press strength were measured before and after RT and compared to databases of younger subjects (n=22-60). Myofibrillar protein degradation (3-methylhistidine) and bone resorption (cross-linked N-telopeptides) were also measured before and after RT. Lean tissue mass, muscle thickness (except ankle dorsi flexors), and strength increased with training (P<0.05), with little difference between groups. There were no changes in 3-methylhistidine or cross-linked N-telopeptides. Before RT, all measures were lower in the older compared to younger groups (P<0.05), except for elbow extensor muscle thickness. Following training, muscle thickness of the elbow flexors and ankle dorsi flexors and leg press strength were no longer different than the young, and elbow extensor muscle thickness was greater in the old men (P<0.05). Supplementation with protein before or after training has no effect on muscle mass and strength in older men. RT was sufficient to overcome deficits in muscle size of the elbow flexors and ankle dorsi flexors and leg press strength in older compared to younger men.

Dietary approach to attenuate oxidative stress, hypertension, and inflammation in the cardiovascular system.

Imbalance between production and scavenging of superoxide anion results in hypertension by the inactivation of nitric oxide, and the increased oxidative stress from the resultant peroxynitrite that is produced promotes inflammatory processes such as atherosclerosis. Induction of phase 2 proteins promotes oxidant scavenging. We hypothesized that intake of dietary phase 2 protein inducers would ameliorate both hypertension and atherosclerotic changes in the spontaneously hypertensive stroke-prone rat. For 5 days/week for 14 weeks, we fed rats 200 mg/day of dried broccoli sprouts that contained glucoraphanin, which is metabolized into the phase 2 protein-inducer sulforaphane (Group A), sprouts in which most of the glucoraphanin was destroyed (Group B), or no sprouts (Group C). After 14 weeks of treatment, no significant differences were seen between rats in Groups B and C. Rats in Group A had significantly decreased oxidative stress in cardiovascular and kidney tissues, as shown by increased glutathione (GSH) content and decreased oxidized GSH, decreased protein nitrosylation, as well as increased GSH reductase and GSH peroxidase activities. Decreased oxidative stress correlated with better endothelial-dependent relaxation of the aorta and significantly lower (20 mm Hg) blood pressure. Tissues from Groups B and C had considerable numbers of infiltrating activated macrophages, indicative of inflammation, whereas animals in Group A had few detectable infiltrating macrophages. There is interest in dietary phase 2 protein inducers as means of reducing cancer incidence. We conclude that a diet containing phase 2 protein inducers also reduces the risk of developing cardiovascular problems of hypertension and atherosclerosis.