Geoffrey W. Krissansen

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 cows 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 cows 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.

β7 integrins contribute to demyelinating disease of the central nervous system

A role for alpha4 integrins in different forms of the multiple sclerosis-like disease experimental autoimmune encephalomyelitis (EAE) has been demonstrated, but the individual contributions of alpha4beta1, alpha4beta7, and the related alphaEbeta7 integrin have not been determined. The P7 integrins alpha4beta7 and alphaEbeta7 play a central role in chronic inflammation, mediating the trafficking, entry, and/or adhesion of lymphocytes in the inflamed pancreas and gut, and their ligands MAdCAM-1, VCAM-1 and E-cadherin are expressed on brain endothelial cells and/or on microvessels in the inflamed central nervous system. Here, we show that an antibody directed against the beta7 subunit greatly attenuates a non-remitting form of EAE, induced by adoptive transfer of myelin oligodendrocyte peptide (MOG35-55)-stimulated T cells. Combinational treatment with both anti-beta7 and alpha4 integrin subunit antibodies led to more rapid and complete remission than that obtained with anti-alpha4 antibody alone, potentially implicating a role for alphaEbeta7 in disease progression. Remission correlated with the down-regulation of the vascular addressins VCAM-1. MAdCAM-1, and ICAM-1 on cerebral blood vessels. Attenuated forms of disease were induced by adoptive transfer of either wild-type encephalitogenic T cells to beta7-deficient gene knockout mice, or of beta7-/-encephalitogenic T cells to wild-type recipients. The former finding indicates that beta7 + ve recruited cells contribute to disease progression. Thus alpha4beta1, alpha4beta7, and alphaEbeta7 integrins may all play a contributory role in the progression of chronic forms of demyelinating disease, and together with their ligands could represent potential targets for improved treatment of some forms of multiple sclerosis.

Opposing effects of arsenic trioxide on hepatocellular carcinomas in mice

Arsenic trioxide (As2O3) is a potent antitumor agent used to treat acute promyelocytic leukemia (APL) and, more recently, solid tumors. However, the dose of As2O3 required to suppress human xenographs in mice is markedly higher than that used to treat APL in humans. Paradoxically, low doses of As2O3 stimulate angiogenesis, which might be expected to promote tumor growth. Clearly, appropriate dosages of As2O3 are required to treat human patients to avoid toxicity and undesirable side effects. In the present study, we investigated As2O3 with respect to its toxicity and effects on tumor growth, angiogenesis and cell apoptosis using H22 hepatocellular carcinoma (HCC) cells in a mouse model of HCC. As2O3 inhibited tumor growth and angiogenesis, and enhanced tumor cell apoptosis at doses greater than 1 mg/kg, but mice lost weight and failed to thrive at doses of 4 mg/kg and greater. In contrast, low doses (<1 mg/kg) of As2O3 promoted tumor growth, upregulated the expression of vascular endothelial growth factor and tumor angiogenesis, and had no effect on tumor cell apoptosis. In vitro studies demonstrated that As2O3 inhibited the proliferation of H22 tumor cells and bovine aortic endothelial cells, and induced their apoptosis in a dose‐ and time‐dependent fashion, suggesting that the mechanism of As2O3‐mediated inhibition of tumor growth is due to direct effects of the drug on both tumor cells and endothelia. In summary, different doses of As2O3 have opposing effects on tumor growth and angiogenesis. The results demonstrate that As2O3 has a narrow window of therapeutic opportunity with respect to dosage, and that low doses of the drug as used in metronomic therapy should be used with extreme caution. (Cancer Sci 2006; 97: 675–681)

TLR2, TLR4 and TLR9 polymorphisms and Crohn's disease in a New Zealand Caucasian cohort.

Background and Aim:  Toll‐like receptors (TLRs) have been identified as susceptibility genes for Crohns 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.

Prevention of a chronic progressive form of experimental autoimmune encephalomyelitis by an antibody against mucosal addressin cell adhesion molecule-1, given early in the course of disease progression

A role for α4 and β7 integrins in mediating leucocyte entry into the central nervous system in the multiple sclerosis (MS)‐like disease experimental autoimmune encephalomyelitis (EAE) has been demonstrated. However, the individual contributions of their respective ligands mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1) and E‐cadherin expressed on the blood–brain barrier has not been determined. In the present paper, it is shown that an antibody directed against MAdCAM‐1, the preferential ligand for α4β7, effectively prevented the development of a progressive, non‐remitting, form of EAE, actively induced by injection of myelin oligodendrocyte glycoprotein peptide (MOG35–55) autoantigen. Combinational treatment with both anti‐MAdCAM‐1, VCAM‐1, and intercellular adhesion molecule‐1 (ICAM‐1) (ligand for integrin lymphocyte function‐associated antigen (LFA)‐1) mAbs led to more rapid remission than that obtained with anti‐MAdCAM‐1 antibody alone. However, neither MAdCAM‐1 monotherapy, nor combinational antibody blockade was preventative when administered late in the course of disease progression. In conclusion, MAdCAM‐1 plays a major contributory role in the progression of chronic EAE and is a potential therapeutic target for the treatment of MS. Critically, antivascular addressin therapy must be given early in the course of disease prior to the establishment of irreversible damage if it is to be effective, as a single treatment modality.

‘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 (∼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 flurouracil, 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 Peyers patches. It increased the production of Th1 and Th2 cytokines within the intestine and tumour, including TNF, IFN‐γ, 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.

Downregulation of nuclear factor-κB p65 subunit by small interfering RNA synergizes with gemcitabine to inhibit the growth of pancreatic cancer

The clinical benefit of gemcitabine for pancreatic cancer is low due to chemoresistance. Nuclear factor (NF)-kappaB, constitutively activated in pancreatic cancer, is a therapeutic target as it upregulates expression of genes controlling proliferation, apoptosis and angiogenesis. This study aimed to investigate whether downregulation of the p65 subunit of NF-kappaB by siRNA could enhance the efficacy of gemcitabine to treat pancreatic cancer. p65 siRNA synergized with gemcitabine to inhibit the proliferation and induce the apoptosis of pancreatic cancer cells in vitro and in vivo, and suppress the growth and angiogenesis of pancreatic tumors in nude mice. The mechanisms involved inhibition of NF-kappaB activity and consequent inhibition of Bcl-2, cyclin D1 and VEGF, and activation of caspase-3. The results suggest that downregulation of NF-kappaB p65 potentiates the efficacy of gemcitabine in combating pancreatic cancer.

Antisense hypoxia‐inducible factor 1α 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‐1α expression vector downregulates expression of HIF‐1α 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‐1α could enhance the therapeutic efficacy of doxorubicin to combat HCC. Both antisense HIF‐1α 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‐1α 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‐1α downregulated the expression of both HIF‐1α and VEGF, whereas doxorubicin only downregulated VEGF expression. Antisense HIF‐1α and doxorubicin synergized to downregulate VEGF expression. Both antisense HIF‐1α and doxorubicin inhibited expression of proliferating cell nuclear antigen, and combined to exert even stronger inhibition of proliferating cell nuclear antigen expression. Antisense HIF‐1α therapy warrants investigation as a therapeutic strategy to enhance the efficacy of doxorubicin for treating HCC. (Cancer Sci 2008; 99: 2055–2061)

Arsenic trioxide synergizes with B7H3-mediated immunotherapy to eradicate hepatocellular carcinomas

Arsenic trioxide (As2O3), a valuable anticancer drug for the treatment of acute promyelocytic leukemia, may also have therapeutic potential for the treatment of solid tumors. However, its therapeutic efficacy against solid tumors is lacking even at high dosages. Other therapeutic strategies are required to enhance the efficacy of As2O3 against solid tumors such as hepatocellular carcinoma (HCC), which is refractory to chemotherapy. B7H3, a new member of the B7 family, has been shown to induce antitumor immunity. Intratumoral injection of B7H3 plasmids eradicates small EL‐4 lymphomas, but monotherapy is ineffective against large tumors. Here we investigated whether As2O3 would synergize with B7H3 immunotherapy to combat HCC. Large subcutaneous H22 HCCs (0.7–0.8 cm in diameter) established in BALB/c mice were rapidly and completely eradicated when intratumoral administration of As2O3 was preceded by in situ gene transfer of B7H3. In contrast, neither As2O3 nor B7H3 monotherapy was effective. The antitumor activity of As2O3 was attributed to increased tumor‐cell apoptosis, perhaps as a result of direct cytotoxicity as well as decreased tumor angiogenesis. Combination therapy generated potent systemic antitumor immunity mediated by CD8+ and NK cells that was effective in combating a systemic challenge of 1 × 107 parental H22 cells. It led to the simultaneous and complete regression of multiple distant tumor nodules, concomitant with increased levels of serum IFN‐γ and cytotoxic T lymphocyte (CTL) activity. In conclusion, combining B7H3‐mediated immunotherapy with As2O3 warrants investigation as a therapeutic strategy to combat HCC, and other malignancies.

Polymorphisms in the organic cation transporter genes SLC22A4 and SLC22A5 and Crohn's disease in a New Zealand Caucasian cohort.

Polymorphisms in the organic cation transporter (OCTN) genes SLC22A4 (OCTN1; polymorphism 1672C/T) and SLC22A5 (OCTN2; polymorphism −207G/C) at the inflammatory bowel disease (IBD) 5 locus comprise a two‐allele haplotype (SLC22A‐TC) associated with increased risk for Crohns disease (CD). In this study, we examined the contribution of the disease susceptibility haplotype SLC22A‐TC to CD in a New Zealand Caucasian population. The frequencies of the gene polymorphisms 1672C/T and −207G/C were examined in 182 patients with CD and 188 ethnically matched controls by PCR‐RFLP analysis. There was a significant difference in the allele frequency (0.444 vs 0.519; P = 0.041) of the 1672T polymorphism in the SLC22A4 gene between controls and patients with CD. In contrast, there was no significant difference (0.497 vs 0.552; P = 0.135) for the −207C polymorphism in the SLC22A5 gene. The homozygote SLC22A‐TC diplotype was significantly associated with an increased risk for CD (odds ratio 2.19), and the SLC22A‐TC haplotype was associated with increased risk (P = 0.0007) of ileocolonic involvement. The population‐attributable risk for the SLC22A‐TC haplotype is 15.1%. Thus, SLC22A‐TC is associated with an increased risk of CD and disease phenotype in our New Zealand CD cohort.