Leukocyte numbers and function in subjects eating n-3 enriched foods: selective depression of natural killer cell levels

Introduction : While consumption of omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) has been recommended for those at risk of inflammatory disease such as rheumatoid arthritis, the mechanism of their anti-inflammatory effect remains to be clearly defined, particularly in relation to the dose and type of n-3 LCPUFA. The objective of this study was to determine whether varying the levels of n-3 LCPUFA in erythrocyte membrane lipids, following dietary supplementation, is associated with altered numbers and function of circulating leukocytes conducive to protection against inflammation. Methods : In a double-blind and placebo-controlled study, 44 healthy subjects aged 23 to 63 years consumed either standard or n-3 LCPUFA-enriched versions of typical processed foods, the latter allowing a target daily consumption of 1 gram n-3 LCPUFA. After six months, peripheral blood leukocyte and subpopulation proportions and numbers were assessed by flow cytometry. Leukocytes were also examined for lymphoproliferation and cytokine production, neutrophil chemotaxis, chemokinesis, bactericidal, adherence and iodination activity. Erythrocytes were analyzed for fatty-acid content. Results : Erythrocyte n-3 LCPUFA levels were higher and absolute leukocyte and lymphocyte numbers were lower in subjects consuming n-3 enriched foods than in controls. There were no changes in the number of neutrophils, monocytes, T cells (CD3+), T-cell subsets (CD4+, CD8+) and B cells (CD19+). However, natural killer (NK) (CD3-CD16+CD56+) cell numbers were lower in n-3 supplemented subjects than in controls and were inversely related to the amount of eicosapentaenoic acid or docosahexaenoic acid in erythrocytes. No significant correlations were found with respect to lymphocyte lymphoproliferation and production of IFN-γ and IL-2, but lymphotoxin production was higher with greater n-3 LCPUFA membrane content. Similarly, neutrophil chemotaxis, chemokinesis, bactericidal activity and adherence did not vary with changes in erythrocyte n-3 LCPUFA levels, but the iodination reaction was reduced with higher n-3 LCPUFA content. Conclusion : The data show that regular long-term consumption of n-3 enriched foods leads to lower numbers of NK cells and neutrophil iodination activity but higher lymphotoxin production by lymphocytes. These changes are consistent with decreased inflammatory reaction and tissue damage seen in patients with inflammatory disorders receiving n-3 LCPUFA supplementation.

Identification of natural killer cell receptor genes in the genome of the marsupial Tasmanian devil (Sarcophilus harrisii)

Within the mammalian immune system, natural killer (NK) cells contribute to the first line of defence against infectious agents and tumours. Their activity is regulated, in part, by cell surface NK cell receptors. NK receptors can be divided into two unrelated, but functionally analogous superfamilies based on the structure of their extracellular ligand-binding domains. Receptors belonging to the C-type lectin superfamily are predominantly encoded in the natural killer complex (NKC), while receptors belonging to the immunoglobulin superfamily are predominantly encoded in the leukocyte receptor complex (LRC). Natural killer cell receptors are emerging as a rapidly evolving gene family which can display significant intra- and interspecific variation. To date, most studies have focused on eutherian mammals, with significantly less known about the evolution of these receptors in marsupials. Here, we describe the identification of 43 immunoglobulin domain-containing LRC genes in the genome of the Tasmanian devil (Sarcophilus harrisii), the largest remaining marsupial carnivore and only the second marsupial species to be studied. We also identify orthologs of NKC genesKLRK1, CD69, CLEC4E, CLEC1B, CLEC1A and an ortholog of an opossum NKC receptor. Characterisation of these regions in a second, distantly related marsupial provides new insights into the dynamic evolutionary histories of these receptors in mammals. Understanding the functional role of these genes is also important for the development of therapeutic agents against Devil Facial Tumour Disease, a contagious cancer that threatens the Tasmanian devil with extinction.

Identification, characterisation and expression analysis of natural killer receptor genes in chlamydia pecorum infected koalas (phascolarctos cinereus)

BACKGROUND: Koalas (Phascolarctos cinereus), an iconic Australian marsupial, are being heavily impacted by the spread of Chlamydia pecorum, an obligate intracellular bacterial pathogen. Koalas vary in their response to this pathogen, with some showing no symptoms, while others suffer severe symptoms leading to infertility, blindness or death. Little is known about the pathology of this disease and the immune response against it in this host. Studies have demonstrated that natural killer (NK) cells, key components of the innate immune system, are involved in the immune response to chlamydial infections in humans. These cells can directly lyse cells infected by intracellular pathogens and their ability to recognise these infected cells is mediated through NK receptors on their surface. These are encoded in two regions of the genome, the leukocyte receptor complex (LRC) and the natural killer complex (NKC). These two families evolve rapidly and different repertoires of genes, which have evolved by gene duplication, are seen in different species. METHODS: In this study we aimed to characterise genes belonging to the NK receptor clusters in the koala by searching available koala transcriptomes using a combination of search methods. We developed a qPCR assay to quantify relative expression of four genes, two encoded within the NK receptor cluster (CLEC1B, CLEC4E) and two known to play a role in NK response to Chalmydia in humans (NCR3, PRF1). RESULTS: We found that the NK receptor repertoire of the koala closely resembles that of the Tasmanian devil, with minimal genes in the NKC, but with lineage specific expansions in the LRC. Additional genes important for NK cell activity, NCR3 and PRF1, were also identified and characterised. In a preliminary study to investigate whether these genes are involved in the koala immune response to infection by its chlamydial pathogen, C. pecorum, we investigated the expression of four genes in koalas with active chlamydia infection, those with past infection and those without infection using qPCR. This analysis revealed that one of these four, CLEC4E, may be upregulated in response to chlamydia infection. CONCLUSION: We have characterised genes of the NKC and LRC in koalas and have discovered evidence that one of these genes may be upregulated in koalas with chlamydia, suggesting that these receptors may play a role in the immune response of koalas to chlamydia infection.

Enhanced cytotoxic function of natural killer and natural killer T-like cells associated with decreased CD94 (Kp43) in the chronic obstructive pulmonary disease airway

Background and objective: Natural killer (NK) and natural killer T (NKT)-like cells represent a small but important proportion of effector lymphocytes that we have previously shown to be major sources of pro-inflammatory cytokines and granzymes. We hypothesized that these cells would be increased in the airway in chronic obstructive pulmonary disease (COPD), accompanied by reduced expression of the inhibitory receptor CD94 (Kp43) and increased expression of cytotoxic mediators granzyme B and perforin.
Methods: We measured NK and NKT-like cells and their expression of CD94 in the blood of COPD patients (n = 71; 30 current and 41 ex-smokers), smokers (16) and healthy controls (25), and bronchoalveolar lavage fluid (BALF) from a cohort of subjects (19 controls, 12 smokers, 33 COPD). Activation was assessed by measuring CD69 in blood and the cytotoxic potential of NK cells by measuring granzymes A and B, and using a cytotoxicity assay in blood and BALF.
Results: In blood in COPD, there were no significant changes in the proportion of NK or NKT-like cells or expression of granzyme A or NK cytotoxic potential versus controls. There was, however, increased expression of granzyme B and decreased expression of CD94 by both cell types versus controls. The proportion of NK and NKT-like cells were increased in BALF in COPD, associated with increased NK cytotoxicity, increased expression of granzyme B and decreased expression of the inhibitory receptor CD94 by both cell types.
Conclusions: Treatment strategies that target NK and NKT-like cells, their cytotoxicity and production of inflammatory mediators in the airway may improve COPD morbidity.

Vascular attack by 5,6-dimethylxanthenone-4-acetic acid combined with B7.1 (CD80)-mediated immunotherapy overcomes immune resistance and leads to the eradication of large tumors and multiple tumor foci

The promise of cancer immunotherapy is that it will not only eradicate primary tumors but will generate systemic antitumor immunity capable of destroying distant metastases. A major problem that must first be surmounted relates to the immune resistance of large tumors. Here we reveal that immune resistance can be overcome by combining immunotherapy with a concerted attack on the tumor vasculature. The functionally related antitumor drugs 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone acetic acid (FAA), which cause tumor vasculature collapse and tumor necrosis, were used to attack the tumor vasculature, whereas the T-cell costimulator B7.1 (CD80), which costimulates T-cell proliferation via the CD28 pathway, was used to stimulate antitumor immunity. The injection of cDNA (60–180 µg) encoding B7.1 into large EL-4 tumors (0.8 cm in diameter) established in C57BL/6 mice, followed 24 h later by i.p. administration of either DMXAA (25 mg/kg) or FAA (300 mg/kg), resulted in complete tumor eradication within 2–6 weeks. In contrast, monotherapies were ineffective. Both vascular attack and B7.1 immunotherapy led to up-regulation of heat shock protein 70 on stressed and dying tumor cells, potentially augmenting immunotherapy. Remarkably, large tumors took on the appearance of a wound that rapidly ameliorated, leaving perfectly healed skin. Combined therapy was mediated by CD8+ T cells and natural killer cells, accompanied by heightened and prolonged antitumor cytolytic activity (P < 0.001), and by a marked increase in tumor cell apoptosis. Cured animals completely rejected a challenge of 1 x 107 parental EL-4 tumor cells but not a challenge of 1 x 104 Lewis lung carcinoma cells, demonstrating that antitumor immunity was tumor specific. Adoptive transfer of 2 x 108 splenocytes from treated mice into recipients bearing established (0.8 cm in diameter) tumors resulted in rapid and complete tumor rejection within 3 weeks. Although DMXAA and B7.1 monotherapies are complicated by a narrow range of effective doses, combined therapy was less dosage dependent. Thus, a broad range of amounts of B7.1 cDNA were effective in combination with 25 mg/kg DMXAA. In contrast, DMXAA, which has a very narrow range of high active doses, was effective at a low dose (18 mg/kg) when administered with a large amount (180 µg) of B7.1 cDNA. Importantly, combinational therapy generated heightened antitumor immunity, such that gene transfer of B7.1 into one tumor, followed by systemic DMXAA treatment, led to the complete rejection of multiple untreated tumor nodules established in the opposing flank. These findings have important implications for the future direction and utility of cancer immunotherapies aimed at harnessing patients’ immune responses to their own tumors.

Effects of 60-day bed rest with and without exercise on cellular and humoral immunological parameters

Exercise at regular intervals is assumed to have a positive effect on immune functions. Conversely, after spaceflight and under simulated weightlessness (e.g., bed rest), immune functions can be suppressed. We aimed to assess the effects of simulated weightlessness (Second Berlin BedRest Study; BBR2-2) on immunological parameters and to investigate the effect of exercise (resistive exercise with and without vibration) on these changes. Twenty-four physically and mentally healthy male volunteers (20-45 years) performed resistive vibration exercise (n=7), resistance exercise without vibration (n=8) or no exercise (n=9) within 60 days of bed rest. Blood samples were taken 2 days before bed rest, on days 19 and 60 of bed rest. Composition of immune cells was analyzed by flow cytometry. Cytokines and neuroendocrine parameters were analyzed by Luminex technology and ELISA/RIA in plasma. General changes over time were identified by paired t-test, and exercise-dependent effects by pairwise repeated measurements (analysis of variance (ANOVA)). With all subjects pooled, the number of granulocytes, natural killer T cells, hematopoietic stem cells and CD45RA and CD25 co-expressing T cells increased and the number of monocytes decreased significantly during the study; the concentration of eotaxin decreased significantly. Different impacts of exercise were seen for lymphocytes, B cells, especially the IgD(+) subpopulation of B cells and the concentrations of IP-10, RANTES and DHEA-S. We conclude that prolonged bed rest significantly impacts immune cell populations and cytokine concentrations. Exercise was able to specifically influence different immunological parameters. In summary, our data fit the hypothesis of immunoprotection by exercise and may point toward even superior effects by resistive vibration exercise.Cellular & Molecular Immunology advance online publication, 10 November 2014; doi:10.1038/cmi.2014.106.

Comparative activities of milk components in reversing chronic colitis

Inflammatory bowel disease (IBD) is a poorly understood chronic immune disorder for which there is no medical cure. Milk and colostrum are rich sources of bioactives with immunomodulatory properties. Here we compared the therapeutic effects of oral delivery of bovine milk-derived iron-saturated lactoferrin (Fe-bLF), angiogenin, osteopontin (OPN), colostrum whey protein, Modulen IBD (Nestle Healthsciences, Rhodes, Australia), and cis-9,trans-11 conjugated linoleic acid (CLA)-enriched milk fat in a mouse model of dextran sulfate-induced colitis. The CLA-enriched milk fat significantly increased mouse body weights after 24d of treatment, reduced epithelium damage, and downregulated the expression of proinflammatory cytokines and nitrous oxide. Modulen IBD most effectively decreased the clinical score at d 12, and Modulen IBD and OPN most effectively lowered the inflammatory score. Myeloperoxidase activity that denotes neutrophil infiltration was significantly lower in mice fed Modulen IBD, OPN, angiogenin, and Fe-bLF. A significant decrease in the numbers of T cells, natural killer cells, dendritic cells, and a significant decrease in cytokine expression were observed in mice fed the treatment diets compared with dextran sulfate administered mice. The Fe-bLF, CLA-enriched milk fat, and Modulen IBD inhibited intestinal angiogenesis. In summary, each of the milk components attenuated IBD in mice, but with differing effectiveness against specific disease parameters.

Anti-cancer natural products inducing cross-talk between apoptosis and autophagy mutual proteins to regulate cancer cell death: design of future green anticancer therapies.

The present letter concerns anti-cancer natural products inducing cross-talk between apoptosis and autophagy mutual proteins to regulate cancer cell death for future cancer green therapeutic approaches. The course of cancer advancement has always been attributed to the defectiveness in cell death mechanisms (Du et al., 2013; Hematulin et al., 2014). These defects act as a shield in protecting tumor cells from drugs and therapies, all at the same time, maintaining a longer life span and prompting their dispersion procedures. Autophagy and apoptosis safeguards cells from cellular damages and maintains proliferation and homeostasis by deporting outgrowth and controlling differentiation of pernicious cells. The autophagic proteins are conventionally found in hindering apoptosis whereas vice versa accounts had been reported for apoptotic-intermediates in preventing autophagic responses.

'Iron-saturated' lactoferrin is a potent natural adjuvant for augmenting cancer chemotherapy

Bovine lactoferrin (bLf), an iron-containing natural defence protein found in bodily secretions, has been reported to inhibit carcinogenesis and the growth of tumours. Here, we investigated whether natural bLf and iron-saturated forms of bLf differ in their ability to augment cancer chemotherapy. bLf was supplemented into the diet of C57BL/6 mice that were subsequently challenged subcutaneously with tumour cells, and treated by chemotherapy. Chemotherapy eradicated large (0.6 cm diameter) EL-4 lymphomas in mice that had been fed iron-saturated bLf (here designated Lf(+)) for 6 weeks prior to chemotherapy, but surprisingly not in mice that were fed lesser iron-saturated forms of bLf, including apo-bLf (4% iron saturated), natural bLf (approximately 15% iron saturated) and 50% iron-saturated bLf. Lf(+)-fed mice bearing either EL-4, Lewis lung carcinoma or B16 melanoma tumours completely rejected their tumours within 3 weeks following a single injection of either paclitaxel, doxorubicin, epirubicin or fluorouracil, whereas mice fed the control diet were resistant to chemotherapy. Lf(+) had to be fed to mice for more than 2 weeks prior to chemotherapy to be wholly effective in eradicating tumours from all mice, suggesting that it acts as a competence factor. It significantly reduced tumour vascularity and blood flow, and increased antitumour cytotoxicity, tumour apoptosis and the infiltration of tumours by leukocytes. Lf(+) bound to the intestinal epithelium and was preferentially taken up within Peyer's patches. It increased the production of Th1 and Th2 cytokines within the intestine and tumour, including TNF, IFN-gamma, as well as nitric oxide that have been reported to sensitize tumours to chemotherapy. Importantly, it restored both red and white peripheral blood cell numbers depleted by chemotherapy, potentially fortifying the mice against cancer. In summary, bLf is a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy, but needs to be saturated with iron to be effective.

Consequences of immunodominant epitope deletion for minor influenza virus-specific CD8+-T-cell responses

The extent to which CD8+ T cells specific for other antigens expand to compensate for the mutational loss of the prominent DbNP366 and DbPA224 epitopes has been investigated using H1N1 and H3N2 influenza A viruses modified by reverse genetics. Significantly increased numbers of CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ T cells were found in the spleen and in the inflammatory population recovered by bronchoalveolar lavage from mice that were first given the -NP-PA H1N1 virus intraperitoneally and then challenged intranasally with the homologous H3N2 virus. The effect was less consistent when this prime-boost protocol was reversed. Also, though the quality of the response measured by cytokine staining showed some evidence of modification when these minor CD8+-T-cell populations were forced to play a more prominent part, the effects were relatively small and no consistent pattern emerged. The magnitude of the enhanced clonal expansion following secondary challenge suggested that the prime-boost with the -NP-PA viruses gave a response overall that was little different in magnitude from that following comparable exposure to the unmanipulated viruses. This was indeed shown to be the case when the total response was measured by ELISPOT analysis with virus-infected cells as stimulators. More surprisingly, the same effect was seen following primary challenge, though individual analysis of the CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ sets gave no indication of compensatory expansion. A possible explanation is that novel, as yet undetected epitopes emerge following primary exposure to the -NP-PA deletion viruses. These findings have implications for both natural infections and vaccines.

Killer T cells in influenza

Antigen-specific CD8+ T cells play an important role in virus clearance. Here we review the current understanding of influenza virus-specific CD8+ T cell immunity in experimental mouse models and humans. The characteristics and nature of CD8+ T cell killing are discussed, as is the selection and maintenance of the influenza-specific effector and memory repertoires. Consideration is given to vaccine strategies and to the effects of ageing. Understanding the complexities of CD8+ T cell mediated immunity and memory has the potential for improving vaccine design, particularly to combat pandemics caused by newly emerging influenza viruses.

No evidence of expression of two classes of natural antibiotics (cathelicidins and defensins) in a sample of platypus milk

Marsupial neonates are born without a fully functioning immune system, and are known to be protected in part by natural antimicrobial peptides present in their mother's milk. Monotreme neonates hatch at a similar stage in development, and it has been hypothesised that their survival in a non-sterile burrow also relies on the presence of natural antibiotics in their mother's milk. Here we review the field of monotreme lactation and the antimicrobial peptide complement of the platypus (Ornithorhynchus anatinus). Using reverse transcriptasepolymerase chain reaction of milk cell RNA from a sample of platypus milk, we found no evidence for the expression of cathelicidins or defensins in the milk. This was unexpected. We hypothesise that these natural antibiotics may instead be produced by the young platypuses themselves.

Lactoferrin and cancer in different cancer models

Lactoferrin (Lf) is a multifunctional protein and an essential element of innate immunity. Cancer is a major killer in today's world accounting for around 13% of all deaths according to the World Health Organisation (W.H.O.). The five most common forms of cancer include lung, colorectal, stomach, liver and breast cancer. Lactoferrin is a natural forming iron-binding glycoprotein with antibacterial, antioxidant and anti-carcinogenic effects. It is produced in exocrine glands and is secreted in many external fluids as a first line of defence. Lactoferrin also has the capacity to induce apoptosis and inhibit proliferation in cancer cells as well as restore white and red blood cell levels after chemotherapy. This review focuses on the therapeutic effect bovine sourced lactoferrin has on various forms of cancer in various models. It also focuses on the benefits of 3D in vitro cell culture. 3D cell culture has vast advantages over 2D models including demonstration of realistic therapeutic results and heightened resistance that 2D models fail to display.

Convection in fluid overlying porous layer : an application to Hall-Heroult cell

Finite-element method is used to predict the buoyancy-driven convection in a horizontal layer of fluid (aluminum melt) overlying a porous layer (cathode) saturated with the same fluid. This work aims to compare the Hall–Héroult process in electrolytic cell, where a layer of molten aluminum is reduced over the porous cathode surface. In this study, the physical system of the aluminum melt (fluid) and cathode (porous) together is considered as a composite system of fluid overlying porous layer. The main objective of this study to analyse the velocity components in thin fluid layer and its impact on a porous cathode surface if there is any. In addition, an externally imposed time-independent uniform magnetic field is used to analyse its influence on natural convective forces. The physical system of fluid overlying porous layer is analysed at different Hartmann, Darcy, and fluid-Rayleigh numbers for a fixed Prandtl number (Pr = 0.014). The predicted data show that the convective forces, caused by buoyancy-driven flow, are significant. It is shown that the velocity peaks moves toward the solid wall because of the presence of a magnetic field creating a stronger boundary-layer growth over the permeable cathode surface. The predicted results are plotted in terms of average Nusselt number and Darcy number to indicate the influence of pores and permeability on overall convective heat-transfer characteristics.

In vitro response to functionalized self-assembled peptide scaffolds for three-dimensional cell culture

Nanomaterials are rich in potential, particularly for the formation of scaffolds that mimic the landscape of the host environment of the cell. This niche arises from the spatial organization of a series of biochemical and biomechanical signals. Self-assembling peptides have emerged as an important tool in the development of functional (bio-)nanomaterials; these simple, easily synthesized subunits form structures which present the properties of these larger, more complex systems. Scaffolds based upon these nanofibrous matrices are promising materials for regenerative medicine as part of a new methodology in scaffold design where a "bottom-up" approach is used in order to simulate the native cellular milieu. Importantly, SAPs hold the potential to be bioactive through the presentation of biochemical and biomechanical signals in a context similar to the natural extracellular matrix, making them ideal targets for providing structural and chemical support in a cellular context. Here, we discuss a new methodology for the presentation of biologically relevant epitopes through their effective presentation on the surface of the nanofibers. Here, we demonstrate that these signals have a direct effect on the viability of cells within a three-dimensional matrix as compared with an unfunctionalized, yet mechanically and morphologically similar system. © 2014 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 197-205, 2014.