Leukocyte integrin α4ß7 associates with heat shock protein 70.

The leukocyte integrin cell adhesion molecules α4ß7 and αEß7 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 ß7 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 ß1, ß2, and ß3 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 ß7 subunit cytoplasmic domain peptide and subsequently identified by high-resolution liquid chromatography-tandem mass spectrometry. In confirmation, the ß7 subunit was co-immunoprecipitated from a T cell lysate by an anti-HSP70 antibody. Further, recombinant human HSP70-1a was precipitated by ß7 cytoplasmic domain-coupled beads. The HSP70 inhibitor KNK437 decreased the expression of HSP70 without affecting the expression of the ß7 integrin. It significantly inhibited α4ß7-mediated adhesion of T cells to mucosal addressin cell adhesion molecule 1 (MAdCAM-1), suggesting HSP70 is critical for maintaining ß7 integrin signalling function. The functional implications of the association of ß7 integrins with the different isoforms of HSP70 warrants further investigation.

Design of a PROTAC that antagonizes and destroys the cancer-forming X-protein of the hepatitis B virus.

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.

X-pep, a novel cell-penetrating peptide motif derived from the hepatitis B virus.

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.

Distribution of insulin mRNA transcripts within the human body.

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.

Downregulation of Skp2 inhibits the growth and metastasis of gastric cancer cells in vitro and in vivo.

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.

"Iron-saturated" bovine lactoferrin improves the chemotherapeutic effects of tamoxifen in the treatment of basal-like breast cancer in mice.

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.

Synthesis and biological evaluation of tyrosine modified analogues of the α4ß7 integrin inhibitor biotin-R8ERY.

The α4ß7 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 ß7 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.

Tyrosine modified analogues of the α4ß7 integrin inhibitor biotin-R8ERY prepared via Click Chemistry: synthesis and biological evaluation.

Our continuing programme aiming at developing inhibitors of integrin α4ß7, 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.

Arabinogalactan proteins contribute to the immunostimulatory properties of New Zealand honeys.

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.

Overexpression of von Hippel-Lindau protein synergizes with doxorubicin to suppress hepatocellular carcinoma in mice.

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.

Hepatic overexpression of heme oxygenase-1 improves liver allograft survival by expanding T regulatory cells.

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-ß, 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-ß, and IL-10 than those of control rats, and the transplanted livers expressed higher levels of Foxp3 and TGF-ß. 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.

Genistein synergizes with arsenic trioxide to suppress human hepatocellular carcinoma.

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.

Mucosal addressin cell-adhesion molecule-1 controls plasma-cell migration and function in the small intestine of mice.

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.

A cell-permeable dominant-negative survivin protein induces apoptosis and sensitizes prostate cancer cells to TNF-α therapy.

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.

Antisense hypoxia-inducible factor 1alpha gene therapy enhances the therapeutic efficacy of doxorubicin to combat hepatocellular carcinoma.

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.

Splicing of NOD2 (CARD15) RNA transcripts.

Mutated variants of NOD2, a cytosolic Toll-like receptor (TLR) that recognizes bacterial peptidoglycan, are responsible for increased susceptibility to Crohn's disease (CD). TLRs and their related plant counterparts, the disease-resistance R proteins, undergo alternative splicing as a means of controlling activity. Here we report that regions of NOD2 RNA transcripts that encode the N-terminal and leucine-rich repeat (LRR) domains are alternatively spliced, potentially creating at least eight putative NOD2 variants. The most common variant is a short truncated isoform designated NOD2-short which terminates at residue position 820 leaving three LRR domains. An N-terminally spliced variant designated NOD2-190 contains only CARD1 and a partial CARD2 domain. The expression of transcripts encoding full-length and alternatively spliced forms of NOD2 was altered in blood mononuclear cells and monocytic cell lines stimulated by bacterial products. NOD2-short and NOD2-190 were inactive and unresponsive to muramyl dipeptide (MDP), but did not antagonize the activity of wild-type NOD2. Alternative splicing of NOD2 transcripts represents a potential mechanism by which the intracellular bacterial sensing activity of NOD2 is altered or down-regulated.

mRNA transfection by a Xentry-protamine cell-penetrating peptide is enhanced by TLR antagonist E6446.

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.

Emerging health properties of whey proteins and their clinical implications.

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.

Correlation of the immunostimulatory activities of honeys with their contents of identified bioactives.

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.

Intramuscular delivery of antiangiogenic genes suppresses secondary metastases after removal of primary tumors.

The success of surgery to remove primary tumors can be compromised by the subsequent outgrowth of metastases. It is recognized that primary tumors secrete antiangiogenic factors that suppress the outgrowth of their daughter metastases. In accord we show here that surgical removal of primary EL-4 lymphomas led to a marked decrease in the levels of circulating angiostatin and endostatin, and promoted the growth of distant nodular tumors. Expression vectors encoding angiostatin and endostatin, formulated with poly-N-vinyl pyrrolidone (PVP), were injected into the tibialis and gastrocnemia muscles, leading to expression of angiostatin and endostatin in muscle fibers. High levels of biologically active exogenous proteins were secreted into the circulation. Intramuscular gene therapy with angiostatin and endostatin plasmids significantly inhibited tumor vascularity and induced tumor cell apoptosis, and thereby suppressed the growth of secondary subcutaneous and disseminated metastatic tumors in the lung and liver. Simultaneous intramuscular delivery of both angiostatin and endostatin plasmids significantly prolonged the survival of mice after removal of primary tumors. These results suggest that intramuscular gene transfer of angiostatin and endostatin might serve as a prophylactic cancer-prevention strategy to combat the recurrence of cancer after surgical resection of primary tumors.