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Messenger RNA (mRNA) transfection is a developing field that has applications in research and gene therapy. Potentially, mRNA transfection can be mediated efficiently by cell-penetrating peptides (CPPs) as they may be modified to target specific tissues. However, whilst CPPs are well-documented to transfect oligonucleotides and plasmids, mRNA transfection by CPPs has barely been explored. Here we report that peptides, including a truncated form of protamine and the same peptide fused to the CPP Xentry (Xentry-protamine; XP), can transfect mRNAs encoding reporter genes into human cells. Further, this transfection is enhanced by the anti-malarial chloroquine (CQ) and the toll-like receptor antagonist E6446 (6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole), with E6446 being >5-fold more potent than CQ at enhancing this transfection. Finally, E6446 facilitated the transfection by XP of mRNA encoding the cystic fibrosis transmembrane regulator, the protein mutated in cystic fibrosis. As such, these findings introduce E6446 as a novel transfection enhancer and may be of practical relevance to researchers seeking to improve the mRNA transfection efficiency of their preferred CPP., Competing Interests: The authors have declared that no competing interests exist.

Honeys with unique compositions and properties are worth studying for their health-promoting effects. In order to correlate bioactive content with immunostimulatory activity we compared the abilities of seventy eight New Zealand and non-New Zealand honeys to stimulate blood monocytes to release tumour necrosis factor (TNF)-α, and examined the compositions of selected honeys that had widely varying activities. All the honeys, except for a Malaysian "Amber honey" stimulated the release of TNF-α from monocytes. However, the honeys differed greatly in their immunostimulatory activity, even within the same honey type. They differed in their contents of immunostimulatory components, including apalbumins, arabinogalactan proteins, and apisimin, whose levels did not correlate exactly with immunostimulatory activities. We suggest that the immunostimulatory properties of honey may be influenced by other factors, including unidentified immunostimulatory bioactives and immunosuppressive components; the bioavailability of some bioactives may depend on unidentified factors., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The leukocyte integrin cell adhesion molecules α4β7 and αEβ7 mediate the homing and retention of lymphocytes to the gut, and sites of inflammation. Here we have identified heat shock protein 70 (HSP70) as a major protein that associates with the cytoplasmic domain of the integrin β7 subunit. HSPs are molecular chaperones that protect cells from stress but more recently have been reported to also regulate cell adhesion and invasion via modulation of β1, β2, and β3 integrins and integrin-associated signalling molecules. Several HSP70 isoforms including HSP70-3, HSP70-1L, HSP70-8, and HSP70-9 were specifically precipitated from T cells by a bead-conjugated β7 subunit cytoplasmic domain peptide and subsequently identified by high-resolution liquid chromatography-tandem mass spectrometry. In confirmation, the β7 subunit was co-immunoprecipitated from a T cell lysate by an anti-HSP70 antibody. Further, recombinant human HSP70-1a was precipitated by β7 cytoplasmic domain-coupled beads. The HSP70 inhibitor KNK437 decreased the expression of HSP70 without affecting the expression of the β7 integrin. It significantly inhibited α4β7-mediated adhesion of T cells to mucosal addressin cell adhesion molecule 1 (MAdCAM-1), suggesting HSP70 is critical for maintaining β7 integrin signalling function. The functional implications of the association of β7 integrins with the different isoforms of HSP70 warrants further investigation.

Background: MicroRNAs (miRNAs) are dysregulated in the inflammatory bowel diseases, Crohn's disease (CD) and ulcerative colitis (UC), which arise due to dysfunctional host-microbe interactions and impairment of the barrier function of the intestine. Here, we sought to determine whether circulating miRNAs are biomarkers of active colonic CD and UC and can provide insights into disease pathogenesis. Comparison was made with serum miRNAs in patients with rheumatoid arthritis (RA)., Methods: Total serum RNA from patients with colonic CD, UC, and RA, and normal healthy adults was screened for disease-associated miRNAs by microarray analysis, with subsequent validation by quantitative reverse-transcription polymerase chain reaction. MiRNA targets were identified by luciferase reporter assays., Results: MiR-595 and miR-1246 were significantly upregulated in the sera of active colonic CD, UC, and RA patients, compared with healthy subjects; and in active colonic CD and UC compared with inactive disease. Luciferase reporter assays indicated that miR-595 inhibits the expression of neural cell adhesion molecule-1 and fibroblast growth factor receptor 2., Conclusions: Serum miR-595 and miR-1246 are biomarkers of active CD, UC, and RA. These findings gain significance from reports that miR-595 impairs epithelial tight junctions, whereas miR-1246 indirectly activates the proinflammatory nuclear factor of activated T cells. miR-595 targets the cell adhesion molecule neural cell adhesion molecule-1, and fibroblast growth factor receptor 2, which plays a key role in the differentiation, protection, and repair of colonic epithelium, and maintenance of tight junctions. miR-595 and miR-1246 warrant testing as potential targets for therapeutic intervention in the treatment of inflammatory bowel disease.

Here we determined whether immunostimulatory plant-derived arabinogalactan proteins (AGPs) and the honeybee-derived protein apisimin are present in varieties of New Zealand honey. Apisimin is a protein of unknown function secreted from the glands of honeybees into Royal Jelly, forming a complex with apalbumin1 capable of stimulating lymphocyte proliferation. AGPs were abundant in kanuka honey with lesser amounts in manuka, kowhai and clover honeys, but absent from Royal Jelly. Apisimin was present in all honeys, as well as Royal Jelly. We report that apisimin shares with honey AGPs the ability to stimulate the release of TNF-α from blood monocytes. Further, it synergizes with AGPs to enhance the release of TNF-α, via a mechanism not involving the formation of a complex with AGPs. In summary, this study provides evidence that AGPs and apisimin are commonly present in different floral varieties of honey, and hence contribute to their immunostimulatory properties., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The X-protein of the hepatitis B virus (HBV) is essential for virus infection and contributes to the development of HBV-induced hepatocellular carcinoma (HCC), a disease which causes more than one million deaths each year. Here we describe the design of a novel PROTAC (proteolysis targeting chimeric molecule) capable of simultaneously inducing the degradation of the X-protein, and antagonizing its function. The PROTAC was constructed by fusing the N-terminal oligomerization and C-terminal instability domains of the X-protein to each other, and rendering them cell-permeable by the inclusion of a polyarginine cell-penetrating peptide (CPP). It was predicted that the oligomerization domain would bind the X-protein, and that the instability domain would cause the X-protein to be targeted for proteasomal degradation. Addition of the PROTAC to HepG2 liver cancer cells, engineered to express full-length and C-terminally truncated forms of the X-protein, resulted in the degradation of both forms of the X-protein. A cell-permeable stand-alone form of the oligomerization domain was taken up by HepG2 cells, and acted as a dominant-negative inhibitor, causing inhibition of X-protein-induced apoptosis. In summary, the PROTAC described here induces the degradation of the X-protein, and antagonizes its function, and warrants investigation in a preclinical study for its ability to prevent or treat HBV infection and/or the development of HCC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cell-penetrating peptides (CPPs) are able to penetrate the plasma membrane and gain access to the interior of any replicating or non-replicating cell, and are being considered as drug delivery agents. Here we describe the serendipitous discovery of a novel CPP motif (MAARLCCQ), designated X-pep, located at the extreme N-terminus of the X-protein of the hepatitis B virus. X-pep, and a C-terminally truncated form of the peptide (MAARL), readily penetrated HepG2 cells. Further truncation by removal of the terminal leucine residue impaired the cell-penetrating activity of peptide, indicating that MAARL is the active core of the peptide. X-pep is located adjacent to another CPP, namely Xentry, and like Xentry is unable to penetrate unactivated resting lymphocytes suggesting selective cell uptake. A D-isomeric form of the MAARL peptide was not cell-permeable, indicating that the cell-penetrating function of the peptide involves stereoselective interaction with a chiral receptor. The discovery of X-pep, which bears no resemblance to known CPPs, allows studies to be undertaken to determine additional characteristics of this novel CPP., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Inflammation is an important and appropriate host response to infection or injury. However, dysregulation of this response, with resulting persistent or inappropriate inflammation, underlies a broad range of pathological processes, from inflammatory dermatoses to type 2 diabetes and cancer. As such, identifying new drugs to suppress inflammation is an area of intense interest. Despite notable successes, there still exists an unmet need for new effective therapeutic approaches to treat inflammation. Traditional drug discovery, including structure-based drug design, have largely fallen short of satisfying this unmet need. With faster development times and reduced safety and pharmacokinetic uncertainty, drug repositioning - the process of finding new uses for existing drugs - is emerging as an alternative strategy to traditional drug design that promises an improved risk-reward trade-off. Using a zebrafish in vivo neutrophil migration assay, we undertook a drug repositioning screen to identify unknown anti-inflammatory activities for known drugs. By interrogating a library of 1280 approved drugs for their ability to suppress the recruitment of neutrophils to tail fin injury, we identified a number of drugs with significant anti-inflammatory activity that have not previously been characterized as general anti-inflammatories. Importantly, we reveal that the ten most potent repositioned drugs from our zebrafish screen displayed conserved anti-inflammatory activity in a mouse model of skin inflammation (atopic dermatitis). This study provides compelling evidence that exploiting the zebrafish as an in vivo drug repositioning platform holds promise as a strategy to reveal new anti-inflammatory activities for existing drugs., (© 2014. Published by The Company of Biologists Ltd.)

Here we sought evidence for the existence of insulin mRNA-producing cells outside the human pancreas. Commercially available complementary DNA (cDNA) arrays prepared from 72 different types of adult human tissues were screened by PCR for transcripts encoding insulin, and other classic pancreatic hormones. Insulin mRNA transcripts were detected by standard PCR in the pancreas, stomach, pylorus region of the stomach, and the duodenum; and additionally by nested PCR in the jejunum, ileum and cecum, but not in other body tissues including the brain and colon. Most of these tissues also variably expressed mRNA transcripts for amylase α2B, amylin, glucagon, somatostatin, and pancreatic polypeptide. In summary, using sensitive PCR methods we have provided evidence for the presence of rare insulin mRNA-expressing cells within the stomach, small intestine, and cecum. Their role at these sites may be to support classical enteroendocrine cells as sentinels to sense and monitor gastric contents passing into and through the bowel., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In addition to satisfying the metabolic demands of cells, mitochondrial metabolism helps regulate immune cell function. To date, such cell-intrinsic metabolic-immunologic cross-talk has only been described operating in cells of the immune system. Here we show that epidermal cells utilize fatty acid β-oxidation to fuel their contribution to the immune response during cutaneous inflammation. By live imaging metabolic and immunological processes within intact zebrafish embryos during cutaneous inflammation, we uncover a mechanism where elevated β-oxidation-fuelled mitochondria-derived reactive oxygen species within epidermal cells helps guide matrix metalloproteinase-driven leukocyte recruitment. This mechanism requires the activity of a zebrafish homologue of the mammalian mitochondrial enzyme, Immunoresponsive gene 1. This study describes the first example of metabolic reprogramming operating within a non-immune cell type to help control its contribution to the immune response. Targeting of this metabolic-immunologic interface within keratinocytes may prove useful in treating inflammatory dermatoses.

Here we describe a structure-function analysis of the cell-penetrating peptide Xentry derived from the X-protein of the hepatitis B virus. Remarkably, the tetrapeptide core LCLR retains the cell-penetrating ability of the parental peptide LCLRPVG, as either an L- or D-enantiomer. Substitution of the cysteine with leucine revealed that the cysteine is essential for activity. In contrast, the C-terminal arginine could be substituted in the L-isomer with lysine, histidine, glutamic acid, glutamine, and asparagine, though the resulting peptides displayed distinct cell-type-specific uptake. Substitution of the leucines in the D-isomer with other hydrophobic residues revealed that leucines are optimal for activity. Surprisingly, linear di- and tetra-peptide forms of Xentry are not cell-permeable. Protease-activatable forms of Xentry were created by fusing Xentry to itself via a protease-cleavable peptide, or by attaching a heparin mimic peptide to the N-terminus. These novel activatable forms of Xentry were only taken up by MCF-7 cells after cleavage by matrix metalloproteinase 9, and could be used to deliver drugs specifically to tumours.

This study investigated the contribution of survivin and its upstream regulators, AKT and hypoxia-inducible factor 1α (HIF-1α), to the resistance of gastric cancer cells to cisplatin (CDDP). We found that over-expression of survivin increased the resistance of SGC7901 and BGC823 gastric cancer cells to CDDP. Its over-expression abrogated CDDP-induced inhibition of cell proliferation and CDDP-induced cell apoptosis. In contrast, down-regulation of survivin expression using small hairpin RNA (shRNA) vectors and the small-molecule inhibitor YM155, or inhibition of survivin function using a recombinant cell-permeable dominant-negative survivin protein (dNSur9), promoted CDDP-induced apoptosis. CDDP-resistant sub-lines generated from the parental SGC7901 and BGC823 cells by exposure to increasing concentrations of CDDP expressed higher levels of HIF-1α and survivin in response to hypoxia, and higher levels of phosphorylated AKT (pAKT). Specific inhibition of AKT reduced the expression of HIF-1α and survivin, whereas specific inhibition or depletion of HIF-1α reduced survivin expression but had no effect on the expression of phosphorylated AKT. The expression levels of survivin affected the therapeutic efficacy of CDDP in treating gastric tumors in mice. Specific inhibition of survivin, AKT and HIF-1α enhanced the sensitivity of CDDP-resistant cells to CDDP. Specific inhibition of survivin, AKT and HIF-1α synergized with CDDP to suppress the growth of gastric tumors that had been engineered to overexpress survivin. In summary, the results provide evidence that up-regulation of survivin by AKT and HIF-1α contributes to CDDP resistance, indicating that inhibition of these pathways may be a potential strategy for overcoming CDDP resistance in the treatment of gastric cancer., (© 2013 FEBS.)

S-phase kinase-associated protein-2 (Skp2) is overexpressed in human cancers and associated with poor prognosis. Skp2 acts as an oncogenic protein by enhancing cancer cell growth and tumor metastasis. The present study has demonstrated that small hairpin RNA (shRNA)-mediated downregulation of Skp2 markedly inhibits the viability, proliferation, colony formation, migration, invasion, and apoptosis of human gastric cancer MGC803 cells, which express a high level of Skp2. In contrast, Skp2 shRNA had only a slight effect on the above properties of BGC823 cells, which express a low level of Skp2. In accord, knockdown of Skp2 suppressed the ability of MGC803 cells to form tumors and to metastasize to the lungs of mice, and the growth of established tumors, by inhibiting cell proliferation and enhancing cell apoptosis. In contrast, overexpression of Skp2 promoted tumorigenesis of BGC823 cells in mice. Skp2 depletion induced cell cycle arrest in the G(1)/S phase by upregulating p27, p21, and p57 and downregulating cyclin E and cyclin-dependent kinase 2. Skp2 depletion also increased caspase-3 activity, impeded the ability of cells to form filopoidia and locomote, upregulated RECK (reversion-inducing cysteine-rich protein with kazal motifs), and downregulated matrix metalloproteinase (MMP)-2 and MMP-9 activity and expression. The results suggest that downregulating Skp2 warrants investigation as a promising strategy to treat gastric cancers that express high levels of Skp2.

Here we describe an entirely new class of cell-penetrating peptide (CPP) represented by the short peptide Xentry (LCLRPVG) derived from an N-terminal region of the X-protein of the hepatitis B virus. Xentry permeates adherent cells using syndecan-4 as a portal for entry, and is uniquely restricted from entering syndecan-deficient, non-adherent cells, such as resting blood cells. Intravenous injection of Xentry alone or conjugated to β-galactosidase led to its delivery to most tissues in mice, except circulating blood cells. There was a predilection for uptake by epithelia. Anti-B-raf antibodies and siRNAs linked to Xentry were capable of killing B-raf-dependent melanoma cells. Xentry represents a new class of CPP with properties that are potentially advantageous for life science and therapeutic applications.

Background: Tamoxifen is used in hormone therapy for estrogen-receptor (ER)-positive breast cancer, but also has chemopreventative effects against ER-negative breast cancers. This study sought to investigate whether oral iron-saturated bovine lactoferrin (Fe-Lf), a natural product which enhances chemotherapy, could improve the chemotherapeutic effects of tamoxifen in the treatment of ER-negative breast cancers., Methods: In a model of breast cancer prevention, female Balb/c mice treated with tamoxifen (5 mg/Kg) were fed an Fe-Lf supplemented diet (5 g/Kg diet) or the base diet. At week 2, 4T1 mammary carcinoma cells were injected into an inguinal mammary fat pad. In a model of breast cancer treatment, tamoxifen treatment was not started until two weeks following tumor cell injection. Tumor growth, metastasis, body weight, and levels of interleukin 18 (IL-18) and interferon γ (IFN-γ) were analyzed., Results: Tamoxifen weakly (IC(50) ~ 8 μM) inhibited the proliferation of 4T1 cells at pharmacological concentrations in vitro. In the tumor prevention study, a Fe-Lf diet in combination with tamoxifen caused a 4 day delay in tumor formation, and significantly inhibited tumor growth and metastasis to the liver and lung by 48, 58, and 66% (all P < 0.001), respectively, compared to untreated controls. The combination therapy was significantly (all P < 0.05) more effective than the respective monotherapies. Oral Fe-Lf attenuated the loss of body weight caused by tamoxifen and cancer cachexia. It prevented tamoxifen-induced reductions in serum levels of IL-18 and IFN-γ, and intestinal cells expressing IL-18 and IFN-γ. It increased the levels of Lf in leukocytes residing in gut-associated lymphoid tissues. B, T and Natural killer (NK) cells containing high levels of Lf were identified in 4T1 tumors, suggesting they had migrated from the intestine. Similar effects of Fe-Lf and tamoxifen on tumor cell viability were seen in the treatment of established tumors., Conclusions: The results indicate that Fe-Lf is a potent natural adjuvant capable of augmenting the chemotherapeutic activity of tamoxifen. It could have application in delaying relapse in tamoxifen-treated breast cancer patients who are at risk of developing ER-negative tumors.

The α4β7 integrin is a well-known target for the development of drugs against various inflammatory disease states including inflammatory bowel disease, type 1 diabetes and multiple sclerosis. The synthesis of a small library of cell-permeable β7 integrin inhibitors based on the peptide biotin-R(8)ERY is reported, in which the tyrosine residue has been modified by using the Suzuki-Miyaura cross-coupling reaction. The synthesised peptidomimetics were evaluated in a cell adhesion assay and shown to inhibit Mn(2+)-activated adhesion of mouse TK-1 T cells to mouse MAdCAM-1. All of the synthesised peptidomimetics are more active than our previously reported lead compound biotin-R(8)ERY with two of the analogues, 6 and 7, exhibiting IC(50) values of <15 μM., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Context: Factors in honey that improve wound healing are poorly understood, but are thought to include lipopolysaccharide (LPS), apalbumin-1 and -2, and a 5.8 kDa component that stimulate cytokine release from macrophages., Objective: To characterize the ability of New Zealand honeys to elicit the release of tumor necrosis factor-α (TNF-α) from monocytic cell lines as a model for early events within a wound site., Materials and Methods: The ability of kanuka (Kunzea ericoides), manuka (Leptospermum scoparium), and clover (Trifolium spp.) honeys to stimulate the release of TNF-α from monocytic cell lines THP-1 and U937 was assayed by ELISA., Results: All three honeys stimulated TNF-α release from THP-1 cells, with kanuka honey being the most active. The activity of kanuka honey was associated with a high molecular weight (>30 kDa) component that was partially heat labile and inhibitable with polymyxin B. LPS concentrations in the honeys were too low to adequately explain the level of immunostimulation. The contribution of type II arabinogalactan proteins (AGPs) we recently identified in kanuka honey was tested, as AGPs are known immunostimulators. AGPs purified from kanuka honey stimulated the release of TNF-α from THP-1 and U937 cells., Discussion: Here we demonstrated that AGPs we recently identified in kanuka honey have immunostimulatory activity. We propose that the immunostimulatory properties of individual honeys relate to their particular content of LPS, apalbumins, the 5.8 kDa component and AGPs., Conclusion: The immunostimulatory activity of kanuka honey may be particularly dependent on AGPs derived from the nectar of kanuka flowers.

Our continuing programme aiming at developing inhibitors of integrin α4β7, a key mediator of various inflammatory diseases, led us to synthesise a library of cell-permeable peptides based on the biotin-R(8)ERY(∗) template, wherein the tyrosine residue has been modified by using the CuAAC reaction. The peptidomimetics were evaluated in a cell adhesion assay and shown to inhibit Mn(2+)-activated adhesion of mouse TK-1 T cells to mouse MAdCAM-1. Two of the synthesised peptidomimetics, analogues 11 and 14, are more active than our previously reported lead compound biotin-r(9)YDRREY at concentrations of 100 and 50 μM, with 14 exhibiting an IC(50) of less than 10 μM., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Blood is collected during animal experimentation to measure haematological and metabolic parameters. It cannot be assumed that circulating blood has the same composition irrespective of its location, and indeed, differences in the composition of blood sampled from the arterial and venous compartments have been reported. Here we investigated whether blood collected by cardiac puncture (CP) versus that collected following removal of the distal 1 mm of the tail tip (TT) differs with respect to glucose and lipid profiles in male C57BL/6J mice at 4, 7, 20 and 28 weeks of age. Blood was first collected from the TT of unanaesthetized mice, which were then immediately anaesthetized using ketamine/xylazine, and a second blood sample was collected by CP. The CP glucose concentration was significantly higher than TT glucose by a positive bias averaging +80% (P < 0.01), irrespective of the age of the mice. Conversely, the concentrations of the CP lipids, including total cholesterol, high-density lipoprotein cholesterol and triglyceride were lower than TT lipids by a negative bias averaging -25% (P < 0.05). These observations highlight the difficulty in measuring and comparing metabolic parameters such as glucose and lipid between one blood compartment and another. They illustrate the need to standardize sampling sites, especially when repeated blood sampling is required.

Heme oxygenase-1 (HO-1) displays anti-inflammatory and cytoprotective activities in sepsis. Here, we investigated the effects of HO-1 on thrombus formation and the protein C system in a septic C57BL/6 mouse model induced by cecal ligation and perforation (CLP). Septic mice were either preinjected with the vehicle, pretreated with hemin (an HO-1 inducer) or zinc protoporphyrin IX (ZnPP, an HO-1 inhibitor), or given a combination of hemin + ZnPP. CLP increased significantly the hepatic expression of HO-1; increased thrombosis in livers, kidneys, and lungs; shortened the prothrombin time (PT) and activated partial thromboplastin time (APTT); elevated the levels of tumor necrosis factor-1α (TNF-1α), interleukin-6 (IL-6), and thrombomodulin (TM); reduced the levels of protein C (PC) and activated protein C (aPC); and downregulated hepatic expression of PC and TM. The preadministration of hemin to septic mice increased the expression and activity of HO-1; inhibited thrombosis in the preceding 3 organs; prolonged PT and APTT; inhibited the production of TNF-α and IL-6; upregulated the expression of PC and TM in livers; elevated the plasma levels of PC and aPC; and reduced the plasma levels of TM. In contrast, ZnPP showed opposite effects to hemin and reversed the effects of hemin by inhibiting the activity of HO-1. The administration of tricarbonyl dichloro ruthenium (II) dimer (CORM-2), which is a CO-releasing molecule, had a similar effect to hemin on thrombosis and the protein C system. The data indicate that the enhanced induction of HO-1 inhibits thrombus formation and affects the protein C system in sepsis., (Copyright © 2012 Mosby, Inc. All rights reserved.)

Milk fat is a natural product containing essential nutrients as well as fatty acids and other food factors with reported anti-cancer potential. Here bovine milk fat was tested for its ability to inhibit the growth of breast and colon cancers and their metastasis to the lung and liver; either alone or in combination with the chemotherapeutic agent paclitaxel. A diet containing 5% typical anhydrous milk fat (representing ~70% of the total dietary fat component) fed to Balb/c mice delayed the appearance of subcutaneous 4T1 breast and CT26 colon cancer tumours and inhibited their metastasis to the lung and liver, when compared to the control diet containing soybean oil as the only fat component. It augmented the inhibitory effects of paclitaxel on tumour growth and metastasis, and reduced the microvessel density of tumours. It displayed no apparent organ toxicity, but instead was beneficial for well-being of tumour-bearing mice by maintaining gastrocnemius muscle and epididymal adipose tissue that were otherwise depleted by cachexia. The milk fat diet ameliorated gut damage caused by paclitaxel in non-tumour-bearing mice, as evidenced by retention of jejunal morphology, villi length and intestinal γ-glutamyl transpeptidase activity, and inhibition of crypt apoptosis. It prevented loss of red and white blood cells due to both cancer-mediated immunosuppression and the cytotoxic effects of chemotherapy. The present study warrants the use of milk fat as an adjuvant to inhibit tumour metastasis during cancer chemotherapy, and to spare patients from the debilitating side-effects of cytotoxic drugs.

Background & Aims: Hypoxia-inducible factors (HIFs) and nuclear factor-κB (NF-κB) regulate genes involved in carcinogenesis and progression of cancers including hepatocellular carcinoma (HCC). The von Hippel-Lindau (VHL) protein (pVHL) targets HIFα subunits for destruction and participates in modulating the activity of NF-κB. The present study aimed to investigate whether the overexpression of pVHL synergizes with doxorubicin in the treatment of HCC., Methods: Overexpression of pVHL was induced by infecting mouse HCC Hepa1-6 and H22 cells, or injecting subcutaneous Hepa1-6 tumors in C57BL/c mice, with adenoviral vectors encoding mouse VHL gene. Cell proliferation, apoptosis, tumoral angiogenesis, and gene expression and DNA-binding activity of NF-κB were examined. The therapeutic effects of pVHL were also evaluated in orthotopic Hepa1-6 tumors by intraportal delivery of Ad-VHL., Results: Ad-VHL enhanced the anti-tumor activity of doxorubicin by inhibiting cell proliferation, and causing cell cycle arrest and apoptosis. The Ad-VHL infection downregulated HIF-1α and HIF-2α expression, and inhibited NF-κB activity and the expression of genes involved in apoptosis, proliferation, angiogenesis, invasion, and metastasis. Injection of Ad-VHL into HCC tumors augmented doxorubicin-induced suppression of tumor growth by inhibiting cell proliferation and tumor angiogenesis, and by inducing cell apoptosis. Effects on the expression of HIFαs, activity of NF-κB, and their downstream genes were in accordance with the in vitro findings. Intraportal injection of Ad-VHL enhanced the efficacy of doxorubicin to suppress the growth of orthotopic liver tumors., Conclusions: By targeting HIF and NF-κB, overexpression of pVHL enhances the efficacy of doxorubicin, and warrants consideration as a potential therapeutic strategy for treating HCC., (Copyright © 2010 European Association for the Study of the Liver. All rights reserved.)

Background: Orally administered milk fat enriched in conjugated linoleic acid (CLA) and trans-vaccenic acid (VA) ('enriched milk fat'), produced by supplementing the diet of pasture-fed cows with fish and sunflower oil, has been shown previously to suppress the development of allergic airway disease in mice., Objective: To investigate whether topical or oral application of enriched milk fat and its two major fatty acids cis-9, trans-11 CLA (c9,t11-CLA) and VA inhibit allergic dermatitis in mice., Methods: Allergic dermatitis was induced in C57BL/6 mice by epicutaneous sensitization of tape-stripped skin with ovalbumin (OVA). Enriched milk fat and its two major fatty acids were either topically applied to the OVA-sensitized skin, or orally fed to mice by supplementation of the diet. Blood and skin tissues were collected for analysis after the third skin sensitization., Results: Both topical and oral administration of enriched milk fat and its two major fatty acids led to significant suppression of allergic dermatitis as evidenced by reduced clinical and histological scores of affected skins, infiltration of inflammatory cells, and circulating allergen-specific IgE levels, compared with treatment with normal milk fat or the base control diet. C9,t11-CLA and VA individually inhibited multiple facets of allergic dermatitis when topically applied, and their combination produced a strong additive effect., Conclusion and Clinical Relevance: Enriched milk fat, and its two major fatty acids c9,t11-CLA and vaccenic acid attenuate allergic dermatitis in mice., (© 2011 Blackwell Publishing Ltd.)

Background: Heme oxygenase (HO)-1 protects transplanted organs from ischemia reperfusion injury and immune rejection. This study sought to investigate whether persistent overexpression of HO-1 in donor livers could improve the survival by expanding T regulatory cells in a rat model of orthotopic liver transplantation., Methods: Livers of Dark Agouti rats were intraportally perfused with an AAV expression vector encoding rat HO-1 (AAV-HO-1), and then transplanted into Lewis rats. The survival, HO-1 activity, Banff rejection activity index, serum levels of IL-2 and TNF-α, infiltration of CD4(+), CD8(+), and T(reg) (CD4(+)CD25(+)Foxp3(+)) cells into donor livers, and expression of Foxp3, TGF-β, and IL-10 were examined. A mixed lymphocyte reaction (MLR) was performed., Results: Intraportal delivery of AAV-HO-1 resulted in persistent expression of HO-1 and increased activity of HO-1 in transplanted livers, leading to prolonged survival of recipients. Overexpression of HO-1 reduced the Banff rejection activity index, and production of IL-2 and TNF-α, inhibited infiltration of CD4(+) and CD8(+) cells, and increased infiltration of T(reg) cells, into donor livers. The spleens of recipients expressed higher levels of Foxp3, TGF-β, and IL-10 than those of control rats, and the transplanted livers expressed higher levels of Foxp3 and TGF-β. Splenocytes from the tolerant recipients had higher percentages of T(reg) cells, and responded poorly to the allogeneic donor splenocytes., Conclusions: Persistent expression of HO-1 in donor livers by intraportal delivery of AAV-HO-1 improves the survival by expanding T(reg) cells. HO-1-based therapies, as described herein, promise new strategies to prevent the rejection of liver transplants., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Survivin is a member of the inhibitor-of-apoptosis (IAP) family which is widely expressed by many different cancers. Overexpression of survivin is associated with drug resistance in cancer cells, and reduced patient survival after chemotherapy and radiotherapy. Agents that antagonize the function of survivin hold promise for treating many forms of cancer. The purpose of this study was to investigate whether a cell-permeable dominant-negative survivin protein would demonstrate bioactivity against prostate and cervical cancer cells grown in three dimensional culture., Results: A dominant-negative survivin (C84A) protein fused to the cell penetrating peptide poly-arginine (R9) was expressed in E. coli and purified by affinity chromatography. Western blot analysis revealed that dNSurR9-C84A penetrated into 3D-cultured HeLa and DU145 cancer cells, and a cell viability assay revealed it induced cancer cell death. It increased the activities of caspase-9 and caspase-3, and rendered DU145 cells sensitive to TNF-α via by a mechanism involving activation of caspase-8., Conclusions: The results demonstrate that antagonism of survivin function triggers the apoptosis of prostate and cervical cancer cells grown in 3D culture. It renders cancer cells sensitive to the proapoptotic affects of TNF-α, suggesting that survivin blocks the extrinsic pathway of apoptosis. Combination of the biologically active dNSurR9-C84A protein or other survivin antagonists with TNF-α therapy warrants consideration as an approach to cancer therapy.

Patients with estrogen receptor-positive (ER+) breast cancers are often treated with aromatase inhibitors or by antiestrogens such as tamoxifen to prevent disease recurrence. Resistant tumors nevertheless develop and it is commonly assumed that they arise by the induction of mutations. However, it is also possible that resistant tumors grow from preexisting variant populations within the original tumor. We have investigated this possibility in the case of the MCF-7 breast cancer cell line. The line was cultured for a prolonged period either in the presence of tamoxifen to block the action of oestrogen or in the absence of estrogen to mimic the action of oophorectomy or treatment with aromatase inhibitors. Both treatments led to growth inhibition followed by eventual outgrowth of sub-lines. Five of these sub-lines were developed and characterized for sensitivity to tamoxifen and to the antibiotic rapamycin, expression of HE R2 and PAX2, and phosphorylation of Akt, p70S6K, 4E-BP1, rpS6, EGFR1, Erk and HE R2. All six lines were ER+ and could be divided into four phenotypes distinguished by cell volume, DNA content (ploidy) and cell cycle time. In two cases, selection with tamoxifen and selection in the absence of estrogen produced similar phenotypes. Rapamycin resistance was a feature of the sub-lines developed under estrogen deprivation and was associated with loss of active phospho-HE R2 and acquisition of PAX2 expression. The results support the conclusion that the MCF-7 cell line is heterogeneous and that the selection conditions allow the growth of pre-existing phenotypes.

Arsenic trioxide (ATO) is of limited therapeutic benefit for the treatment of solid tumors. Genistein exhibits anticancer and pro-oxidant activities, making it a potential candidate to enhance the efficacy of ATO whose cytotoxicity is oxidation-sensitive. This study sought to determine whether genistein synergizes with ATO to combat hepatocellular carcinoma (HCC). Three human HCC cell lines, namely HepG2, Hep3B, and SK-Hep-1, were incubated with ATO, genistein, or ATO + genistein. The cells were also pretreated with antioxidant agents N-acetyl-L-cysteine (NAC) or butylated hydroxyanisole (BHA). Cell viability, apoptosis, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (DeltaPsim), expression of Bcl-2, Bax, caspase-9, and -3, and release of cytochrome c into the cytosol were examined. The synergistic effect of ATO and genistein was also assessed using HepG2 xenografts subcutaneously established in BALB/c nude mice. The results show that genistein synergized with ATO to reduce viability, induce apoptosis, and diminish the DeltaPsim of cells. The combination therapy down-regulated Bcl-2 expression, up-regulated Bax expression, enhanced the activation of caspase-9 and -3, and increased the release of cytochrome c. The synergistic effect of ATO and genistein was diminished by pretreatment with NAC or BHA. Genistein increased the production of intracellular ROS, while ATO had little effect. Genistein synergized with a low dose of ATO (2.5 mg/kg) to significantly inhibit the growth of HepG2 tumors, and suppress cell proliferation and induce apoptosis in situ. There were no obvious side effects, as seen with a high dose of ATO (5 mg/kg). Combining genistein with ATO warrants investigation as a therapeutic strategy to combat HCC.

This study aims to investigate the role of stromal cell-derived factor 1alpha (SDF-1alpha) and its receptor CXCR4 in cellular infiltration of the lung in differentiation syndrome (DS). The acute promyelocytic leukemia (APL) NB4 cells and freshly prepared APL cells from the patients were differentiated by all-trans retinoic acid (ATRA). The expression of SDF-1alpha in human lung tissues was examined by RT-PCR and Western blot analysis. The cells were subjected to adhesion, migration or invasion assays, and co-cultured with human lung tissues in a microgravity rotary cell culture system to examine cellular infiltration in situ. ATRA-differentiated cells expressed high levels of CXCR4, and adhered more strongly to matrigel. Their ability to migrate and invade was enhanced by SDF-1alpha and lung homogenate, and diminished by pre-treatment with an anti-CXCR4 blocking antibody. SDF-1alpha was expressed in the lung tissues of all seven human donors. ATRA-differentiated NB4 cells infiltrated into lung tissues, and this was reduced by pre-treatment with an anti-CXCR4 blocking antibody. The interaction of SDF-1alpha and CXCR4 plays an important role in pulmonary cellular infiltration during DS, suggesting that targeting SDF-1alpha and CXCR4 may provide the basis for potential treatments in the management of DS.

Background & Aims: Immunoglobulin (Ig) A secretion into the intestinal lumen is an important immune mechanism of the gastrointestinal (GI) tract. B cells proliferate and differentiate into IgA-secreting plasma cells (PC) within lymphoid organs then migrate directly into the intestinal lamina propria. We aimed to elucidate the in vivo role of the mucosal addressin cell-adhesion molecule-1 (MAdCAM-1), which is constitutively expressed in the GI-associated lymphoid tissue, in B-cell migration., Methods: We generated MAdCAM-1-deficient mice (MAdCAM(Delta)) and evaluated the B-cell compartment of the GI-associated lymphoid tissue. We also assessed PC migration to the small intestine and the intestinal immune response after oral immunization., Results: In MAdCAM(Delta) mice, the size of Peyer's patches was drastically reduced, compared with that of wild-type mice; this difference was detectable by 3 days after birth, indicating that MAdCAM-1 is dispensable for embryonic Peyer's patch development but mediates recruitment of lymphocytes into this lymphoid organ at later stages. Moreover, antigen-specific, IgA-positive PC were severely compromised in their migration to the small intestine; accordingly, there was a reduced number of IgA-secreting PC in the lamina propria of the small intestine. The MAdCAM(Delta) mice were unable to mount a normal intestinal IgA response after oral immunization with cholera toxin., Conclusion: These data provide in vivo evidence that MAdCAM-1 is required for the localization and function of IgA-secreting PC in the intestine.

Objectives: This study investigated the role of hypoxia-inducible factor 1alpha (HIF-1alpha) in acute pancreatitis (AP) and whether HIF-1alpha is involved in the therapeutic effects of hyperbaric oxygen (HBO) on AP., Methods: Thirty Wistar rats with taurocholate-induced AP were randomly assigned to 3 groups (each group had 10 rats) receiving oxygen, HBO, or no therapeutic treatment 4 hours after induction. Ten healthy sham-operated rats also served as controls. The arterial oxygen saturation, PaO2, pH, lactate dehydrogenase in the arterial sera, and amylase and tumor necrosis factor alpha in the venous sera were measured 6 hours after induction. Pancreatic tissues were subjected to histopathologic analysis, immunohistochemical and Western-blotted analyses of HIF-1alpha and vascular endothelial growth factor, and measuring of myeloperoxidase activity., Results: The HBO therapy attenuated the severity of acute pancreatitis; reduced histopathologic scores, dry weight-wet weight ratio of pancreatic tissues, and levels of amylase and lactate dehydrogenase; and elevated blood arterial oxygen saturation, PaO2, and pH values. The HBO therapy inhibited AP-induced up-regulation of HIF-1alpha and its downstream effector vascular endothelial growth factor and the production of tumor necrosis factor alpha and myeloperoxidase activity., Conclusions: Hypoxia-inducible factor 1alpha plays a key role in the pathogenesis of AP, and the ability to down-regulate the expression of HIF-1alpha may partially explain the therapeutic effect of HBO on AP.

Transcatheter arterial embolization (TAE) is a standard treatment for unresectable hepatic malignancies. It blocks the arterial blood supply to the tumor, but blockade of the blood supply can be short-lived as collateral blood vessels develop, leading to the failure of TAE. Here we report that intraportal delivery of adeno-associated viral (AAV) vectors expressing antisense hypoxia-inducible factor-1alpha (HIF-1alpha) (AAV-ASHIF) augments TAE to combat hepatocellular carcinoma (HCC). Intraportal delivery of AAV-ASHIF led to long-term localized expression of transgenic ASHIF in rat liver, and suppressed the growth of CBRH7919 HCC tumors established in rat liver by inhibiting the formation of neovessels and tumor cell proliferation. TAE therapy caused the necrosis and shrinkage of liver tumors; however, neovessels quickly formed and the residual tumors underwent rapid expansion. TAE enhanced tumor and liver hypoxia, which in turn upregulated expression of HIF-1alpha, vascular endothelial growth factor, glucose transporter-1, lactate dehydrogenase A, and proliferating cell nuclear antigen. Intraportal injection of AAV-ASHIF augmented the therapeutic effects of TAE and diminished its undesirable effects, resulting in extensive tumor cell death and suppression of the growth of liver tumors. In conclusion, this study has revealed that HIF-1 impedes the response of liver tumors to TAE. Antisense HIF-1alpha therapy is warranted as an approach for enhancing the efficacy of TAE to treat unresectable liver cancers.

CARD15 genes carrying the 3020insC frameshift polymorphism encode a truncated CARD15 protein that is unresponsive to bacterial muramyl dipeptide, and are strongly associated with increased susceptibility to Crohn's disease (CD). In this study we established that CARD15 gene sequences encompassing the major 3020insC polymorphism could be readily amplified from the DNA found in saliva. In addition, CARD15 RNA sequences can be readily derived from the cellular component of saliva, which is primarily comprised of buccal epithelial cells. Our results demonstrate that saliva is a readily accessible source of DNA and RNA for genotyping CD patients for variants of the CARD15 gene, representing an alternative source of nucleic acid to that obtained from venous blood.

Hepatocellular carcinoma (HCC), one of the most common cancers worldwide, is resistant to anticancer drugs. Hypoxia is a major cause of tumor resistance to chemotherapy, and hypoxia-inducible factor (HIF)-1 is a key transcription factor in hypoxic responses. We have previously demonstrated that gene transfer of an antisense HIF-1alpha expression vector downregulates expression of HIF-1alpha and vascular endothelial growth factor (VEGF), and synergizes with immunotherapy to eradicate lymphomas. The aim of the present study was to determine whether gene transfer of antisense HIF-1alpha could enhance the therapeutic efficacy of doxorubicin to combat HCC. Both antisense HIF-1alpha therapy and doxorubicin suppressed the growth of subcutaneous human HepG2 tumors established in BALB/c nude mice, tumor angiogenesis, and cell proliferation, and induced tumor cell apoptosis. The combination therapy with antisense HIF-1alpha and doxorubicin was more effective in suppressing tumor growth, angiogenesis, and cell proliferation, and inducing cell apoptosis than the respective monotherapies. Gene transfer of antisense HIF-1alpha downregulated the expression of both HIF-1alpha and VEGF, whereas doxorubicin only downregulated VEGF expression. Antisense HIF-1alpha and doxorubicin synergized to downregulate VEGF expression. Both antisense HIF-1alpha and doxorubicin inhibited expression of proliferating cell nuclear antigen, and combined to exert even stronger inhibition of proliferating cell nuclear antigen expression. Antisense HIF-1alpha therapy warrants investigation as a therapeutic strategy to enhance the efficacy of doxorubicin for treating HCC.

Expression of the canine 180-kDa ribosome receptor p180 in yeast induces the synthesis of RER, and increases the mRNAs of secretory pathway proteins, and protein secretion. To assess whether p180 is a master regulator of cell secretion in mammalian cells, we stably expressed red fluorescent forms of the human p180 variants p180DeltaR (no tandem repeats), p180R (26 repeats), and full-length p180FR (54 repeats) containing different lengths of the tandem repeat ribosome-binding domain in rat pancreatic RINm5F islet beta-cells. All three fluorescent p180 variants localized exclusively to the RER. Cells transfected with p180R were filled with ribosome-studded karmellae, whereas p180DeltaR and p180FR transfectants contained only increased amounts of mostly smooth ER. Unlike in yeast, over-expression of p180R failed to increase the secretory pathway proteins calnexin, SEC61beta, and calreticulin, or ribosome biogenesis. The data suggest that alternative splicing of the p180 tandem repeat domain is a means of regulating the ribosome-binding activity of p180, and potentially the secretory activity of the cell. However, p180 is not a master regulator of mammalian cell secretion as it does not concomitantly trigger the synthesis of protein machinery required to enhance protein synthesis and cell secretion.

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.

Chemotherapy combined with antiangiogenic therapy is more effective than chemotherapy alone. The aim of this study was to investigate whether endostatin, a potent anti-angiogenic agent, could enhance the efficacy of paclitaxel to combat breast cancer. An expression plasmid encoding mouse endostatin (End-pcDNA3.1) was constructed, which produced intense expression of endostatin and inhibited angiogenesis in the chorioallantoic membrane assay. 4T1 breast tumors were established in BALB/c mice by subcutaneous injection of 1 x 10(5) 4T1 cells. The End-pcDNA3.1 plasmid diluted in the transfection reagent FuGENE was injected into the tumors (around 100 mm(2)), and paclitaxel was injected i.p. into the mice. Endostatin gene therapy synergized with paclitaxel in suppressing the growth of 4T1 tumors and their metastasis to the lung and liver. Both endostatin and paclitaxel inhibited tumor angiogenesis and induced cell apoptosis. Despite the finding that endostatin was superior to paclitaxel at inhibiting tumor angiogenesis, paclitaxel was nevertheless more effective at inducing tumor apoptosis. The combination of paclitaxel and endostatin was more effective in suppressing tumor growth, metastases, angiogenesis, and inducing apoptosis than the respective monotherapies. The combinational therapy with endostatin and paclitaxel warrants future investigation as a therapeutic strategy to combat breast cancer.

Background: It has been argued that a reduction in the Western diet of anti-inflammatory unsaturated lipids, such as n-3 polyunsaturated fatty acids, has contributed to the increase in the frequency and severity of allergic diseases., Objective: We investigated whether feeding milk fat enriched in conjugated linoleic acid and vaccenic acids (VAs) ('enriched' milk fat), produced by supplementing the diet of pasture-fed cows with fish and sunflower oil, will prevent development of allergic airway responses., Methods: C57BL/6 mice were fed a control diet containing soybean oil and diets supplemented with milk lipids. They were sensitized by intraperitoneal injection of ovalbumin (OVA) on days 14 and 28, and challenged intranasally with OVA on day 42. Bronchoalveolar lavage fluid, lung tissues and serum samples were collected 6 days after the intranasal challenge., Results: Feeding of enriched milk fat led to marked suppression of airway inflammation as evidenced by reductions in eosinophilia and lymphocytosis in the airways, compared with feeding of normal milk fat and control diet. Enriched milk fat significantly reduced circulating allergen-specific IgE and IgG1 levels, together with reductions in bronchoalveolar lavage fluid of IL-5 and CCL11. Treatment significantly inhibited changes in the airway including airway epithelial cell hypertrophy, goblet cell metaplasia and mucus hypersecretion. The two major components of enriched milk fat, cis-9, trans-11 conjugated linoleic acid and VA, inhibited airway inflammation when fed together to mice, whereas alone they were not effective., Conclusion: Milk fat enriched in conjugated linoleic and VAs suppresses inflammation and changes to the airways in an animal model of allergic airway disease.

The nursery rhyme "Little Miss Muffet sat on a tuffet (small stool) eating her curds and whey. ..." is recognition of the fact that over the centuries "curds and whey", the two major components of cow's milk, have been widely accepted as part of a healthy diet. Milk provides complete nourishment for the neonate for six months from birth, containing factors that help develop various organ systems including the brain, immune system, and the intestine. Importantly it provides immune protection at a time when the neonates own immune system, though fully developed, is albeit immature. Many adult consumers include cow's milk as part of a healthy diet as it provides protein and essential nutrients, vitamins, and minerals, in particular calcium for strong bones. There is a growing appreciation that milk, and in particular whey, contains components that not only provide nutrition, but can also prevent and attenuate disease, or augment conventional therapies, when delivered in amounts that exceed normal dietary intakes. This paper reviews the emerging health properties of whey proteins and their clinical implications.

Background and Aim: Toll-like receptors (TLRs) have been identified as susceptibility genes for Crohn's disease (CD) in some, but not all, studies. Here we examined the association between candidate disease-susceptibility polymorphisms in the TLR2, TLR4 and TLR9 genes and CD in a New Zealand Caucasian population., Methods: The frequency of gene polymorphisms was examined in 182 CD patients and in 188 ethnically matched controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis., Results: We could not detect any significant difference in the allele frequencies of polymorphisms in the TLR2 (R753Q, 0.029 vs 0.016, P = 0.25), TLR4 (D299G and T399I, 0.085 vs 0.071, P = 0.49; and 0.085 vs 0.082, P = 0.90), and TLR9 (-1237T/C, 0.154 vs 0.148, P = 0.82) genes between controls and patients, respectively. There was no evidence that the variant TLR alleles were associated with disease phenotype. However, combination of the datasets of published studies with our dataset confirmed that the TLR4 polymorphism 299G (P = 0.0005; OR of 1.42 [95% CI 1.17-1.74]) and the TLR9 polymorphism -1237C (P = 0.0416; OR of 1.33 [95% CI 1.01-1.75]) are associated with CD., Conclusions: There was no evidence that the above variants of the TLR2, TLR4 and TLR9 genes are major risk factors for CD or influence disease phenotype in our New Zealand case-control study. Nevertheless, the significance of the TLR4 299G and TLR9-1237C associations with CD worldwide was confirmed by a meta-analysis test using our datasets and datasets from previously published studies.

Background and Aim: Crohn's disease (CD) is a complex polygenetic inflammatory disease of the bowel. NFKBIA (IkappaBalpha) is a downstream regulator of NF-kappaB, which is an effector of the major Crohn's susceptibility gene CARD15/NOD2. ICAM-1 is an integrin ligand and vascular addressin, which contributes to the trafficking of pathogenic leukocytes into the inflamed bowel. Here we examined whether the NFKBIA 2758G/A and ICAM-1 K469E variants, recently shown to be CD susceptibility genes in Caucasian populations in Germany and the United Kingdom, are associated with CD in Caucasian patients in New Zealand., Methods: PCR-RFLP analysis was used to examine the frequency of gene polymorphisms in 182 CD patients and 188 ethnically matched controls., Results: There was no significant evidence of a difference in the allele frequency of either the 2758G/A polymorphism in NFKBIA (0.38 vs 0.35, P = 0.47) or the K469E polymorphism in ICAM-1 (0.43 vs 0.37, P = 0.10) between CD patients and controls. Nevertheless, the ICAM-1 469E allele significantly (P = 0.016, Pc = 0.047) influenced the age of diagnosis of CD. Meta-analysis with five other studies confirmed a lack of association of the K469E polymorphism with Crohn's disease (P = 0.33; OR = 0.93; 95% CI, 0.82-1.07)., Conclusion: The NFKBIA 2758G/A and ICAM-1 K469E polymorphisms are not significant risk factors for CD in New Zealand; however, there is evidence that the latter polymorphism influences the age of diagnosis.