Immunity science gets fortified

When it comes to immunomodulation, 2005 witnessed some compelling research that launched new ingredients, reinvigorated older ingredients, and, in one big study, cast aspersions on an old standby. Mark Tallon, PhD, explores

The normal functioning of our immune system is vital for optimal health and physiological function. With the 2003 outbreak of SARS, increased stress-related illnesses in the workplace, and growing pollution and antibiotic resistance, the function and maintenance of the immune system has become a hot issue on everyone's lips. This increase in public awareness of immune control has opened the door for phenomenal growth for innovative immune-boosting dietary supplements.

Of the many factors that modulate immune system function, dietary intake and its specific food components can bring about greater immune responsiveness to viral, bacterial and pathogenic challenges. The result of this enhanced immune response can be improved health, a reduced viral load and reduced disease risk.1

Although many products claim to be immune system boosters, very few have sufficient data behind them to back up these claims. In fact, assessing diet-induced changes of immune function requires targeting a large spectrum of immune system-related diseases, and subsequently the outcome and specific ranges of biomarkers. However, based on a current review of the literature, no single marker allows conclusions to be drawn about the modulation of the whole immune system, except for the clinical outcome of infection itself.1

Therefore, although this article will summarise some of the most influential developments in immuno-enhancing supplementation, be aware that combining biomarkers with high and medium suitability is currently the best approach to measure immunomodulation in human nutrition outside of clinical outcome, which is rarely assessed.

CLA and fatty acids
In recent years, the potential application of the immunomodulatory effects of polyunsaturated fatty acids (PUFAs) in a variety of inflammatory disorders has been of considerable interest to the dietary supplements industry. Dietary PUFAs and particularly those of omega-3 (n-3) and omega-6 (n-6) origin have a variety of anti-inflammatory and immune-modulating effects that may be of relevance to arteriosclerosis, myocardial infarction, stroke and sudden death.2

More recently though, conjugated linoleic acid (CLA) is one or more of eight possible twisted trans fatty acids created from linoleic acid, also known as an n-6 essential fatty acid (EFA). Its promotion as a fat-loss agent has overshadowed its use in immunity, but recent evidence is beginning to shed a light on this hazy zone. These n-3 and n-6 fatty acids appear to be the most potent of fatty acids when it comes to immunomodulation, and as such 2005 has seen some new work in these areas.

According to Doris Bell, scientist for Cognis' Tonalin brand CLA in Germany, "CLA shows tremendous promise in the area of immunity. While the majority of published clinical studies on CLA have focused on reducing body fat and maintaining lean muscle, animal studies show that CLA may play a powerful role in immunity. Initial data in humans raise hope that CLA may have effects similar to those observed in animals.

"Specifically, CLA appears to enhance cell-mediated immune defence reactions, boost immune functions especially for those who are low responders to vaccinations, and provide anti-inflammatory properties for those with asthma and probably other inflammation-based conditions," Bell said. Some studies note CLA levels may raise C-reactive protein, an inflammatory marker, while Bell notes that a study to be published next month in FASEB Journal concludes CLA intervention to have no effect on CRP levels.

Researchers from the renowned Rowett Institute, Scotland, investigated the effects of 12 weeks of CLA supplementation (3g/day: 50% CLA 9-11 and 50% CLA 10-12) on the immune system in healthy humans. The results of the study showed CLA supplementation decreased the levels of pro-inflammatory cytokines (TNF-alpha and IL-1beta) but increased the levels of the anti-inflammatory cytokine, IL-10. This shows a trend toward positive benefits of CLA on the immune system, which may beneficially affect immune function in healthy human volunteers.

Although this data is positive, more recent evidence shows little impact on human markers of immune function.4 These differences in outcome are worthy of further investigation to truly assess if CLA is an effective immunomodulator.

Researchers at the Department of Clinical Pharmacy and Pharmacology, Kagoshima University, Japan, recently investigated the influence of pre-operative (cancer surgery) immune and inflammatory response following administration of a supplement rich in arginine (12.8/l), omega-3 fatty acids (EPA = 2G DHA = 1.4g/l) and RNA (1.29/l).⁵ Patients in the supplements group (n = 12) received one l/day of the supplement for five days before surgery, whilst those in the control group (n = 14) received an ordinary diet without supplementation before surgery. Plasma levels of omega-3 and omega-6 fatty acids, thromboxane B(2), prostaglandin E(2), inflammatory markers, nutritional markers, cytokines and cytokine receptors were obtained five days before the operation at the starting point of supplementation in the supplements group. Samples were collected on post-operative days (PODs) including days 0, 1, 3 and 7.

After taking the supplement, significant increases in omega-3 fatty acids and rapid turnover proteins were found the day after ending supplementation (POD-0), whereas thromboxane B(2) levels and the ratio of omega-6 fatty acids to omega-3 fatty acids were significantly lower than before supplementation. On POD-0 only, inflammatory markers and cytokine receptors in the supplements group showed low levels in comparison with the control group. On POD-1 and POD-3, remarkable decreases in polymorphonuclear leukocyte-elastase and interleukin-8 in the supplements group were observed. These findings suggest that oral administration of a supplement rich in omega-3 fatty acids for five days before surgery may improve not only pre-operative nutritional status but also pre-operative and post-operative inflammatory and immune responses in patients who have cancer.

Beta-glucans
Beta-glucans are a heterogeneous group of glucose polymers found in fungi, plants and some bacteria. These fungal extracts have been known to modulate immune function for millennia, but interest over the last few decades has focused particularly on beta-glucans. The administration of purified beta-glucans has been shown to have a number of beneficial effects, including protection against infections with fungal, bacterial, viral and protozoal pathogens.6 As such, this firm foundation paved the way for beta-glucan development for use in the dietary supplements sector.

"Oat beta-glucan has long been recognised as a topical immuno-stimulant and has been extensively used as an anti-inflammatory agent in skin-care products," said Richard Potter, PhD, chief science officer of Nurture. "More recently, oat beta-glucan has been investigated orally and parenterally as a means of enhancing resistance to bacterial and parasitic infections, and this has shown considerable promise in animal models."7^,8

This is exemplified by a series of animal studies at the University of South Carolina using concentrated oat beta-glucan (OatVantage brand from Nurture Inc).9^,10,11^,12 These studies demonstrated significantly improved morbidity, mortality and resistance to herpes simplex virus (HSV-1) with oat beta-glucan; also, lung tumour metastasis was significantly reduced.

It is believed this is generally related to the affinity of the beta-(1-3) linkage toward specific macrophage receptors. Since the same receptors occur in humans, there is potential for developing this treatment as a countermeasure against the increasing occurrence of microbial resistance to antibiotics and other widely used pharmaceuticals.

Recent work has investigated the effects of beta-glucan on immune function in weaned piglets.¹³ Pigs were fed a diet without or with supplemented beta-glucan (50mg/kg feed). All pigs were injected with ovalbumin (OVA) on day 14 to investigate their humoral immune response (OVA is used as a well-defined antigen bringing about well-studied immune responses).

On day 28, lymphocytes were isolated from all pigs to determine the effects of beta-glucan on cellular immunity of pigs in vitro. Lymphocytes from six pigs of each group were incubated with lipopolysaccharide (LPS). Samples were collected at 0, 3, 6, 12, 18, 24 and 48 hours after LPS addition for determination of interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10).

On day 31, six pigs of each group were injected with either LPS (25 microg/kg body weight) or an equivalent amount of sterile saline. Blood samples were collected at three hours after LPS injection for analysis of IL-6, TNF-alpha and IL-10 in plasma.

The results indicate that dietary beta-glucan enhanced pig antibody response to OVA only in the first week after injection. In vitro, the increases of IL-6 and TNF-alpha in culture medium were partially dampened in pigs supplemented with beta-glucan when their lymphocytes were incubated with LPS. In vivo, dietary beta-glucan attenuated the increase of plasma IL-6 and TNF-alpha, and enhanced the increase of plasma IL-10 when pigs were challenged with LPS.

This evidence further demonstrates that beta-glucans can improve the humoral immunity of pigs and modulate cellular immunity of pigs by mitigating the elevation of pro-inflammatory cytokines and enhancing the increase of anti-inflammatory cytokines after an immunological challenge.

Lactoferrin and whey
Lactoferrin is a non-heme iron-binding glycoprotein produced during lactation and by epithelial cells at mucosal surfaces. The protein is a prominent component of the first line of host defence, and its expression is upregulated in response to inflammatory stimulation.

The results of recent pre-clinical and clinical studies demonstrating that lactoferrin acts as an inhibitor of dermal inflammatory cytokine production have been summarised.14 Lactoferrin seems to act as an anti-inflammatory protein at local sites of inflammation, including the respiratory and gastrointestinal tracts. From the looks of recent work, this seems to have been further corroborated.

Experimental evidence from previous studies supports the conclusion that orally administered lactoferrin restores the immune response in mice treated with a sub-lethal dose of cyclophosphamide, an antineoplastic agent that prevents cancerous growth.15 The aim of a study carried out by researchers at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences was to elucidate potential benefits of lactoferrin in mice undergoing chemotherapy with Busulfan and Cyclophosphamide, followed by intravenous injection of bone marrow cells. Busulfan, like cyclophosphamide, is an antineoplastic medication.

Mice were treated orally with Busulfan (4mg/kg) for four consecutive days, followed by two daily doses of Cyclophasphamide delivered intraperitoneally at a dose of 100mg/kg and reconstituted the next day an injection of syngenetic bone marrow cells. One group of these mice was given lactoferrin in drinking water (0.5 per cent solution). After treatment, mice were immunised with ovalbumin to subsequently determine humoral immunity by evaluation of splenic antibody-forming cells.

Humoral and cellular immune responses of mice treated with chemotherapeutic agents were markedly impaired. This impairment was attenuated by oral administration of lactoferrin, with humoral immunity falling to levels that were 66-88 per cent lower than those that were untreated. Humoral immunity of lactoferrin-treated animals was equivalent to that of untreated mice within one month. Cellular immune responses were inhibited by chemotherapy treatment to a lesser degree, reaching levels that were approximately 50 per cent lower than those of untreated animals.15

In summary, this study suggests that lactoferrin may be useful in accelerating the restoration of immune responsiveness induced by chemotherapy in bone marrow transplant recipients.

The state of the science with whey protein on immune responses is growing. "There is ample evidence from human trials to demonstrate that whey, which typically contains about 50 per cent beta-lactoglobulin, increases glutathione status in white blood cells,"16^,17 said nutritional and protein biochemist Robert Child, PhD, CEO of Alimentarious, a UK-based consultancy. "In immune-compromised subjects, this can translate to increased cytokine production, natural killer cell activity and lower incidence of infections.18^,19 These endpoints are likely to reflect genuine immune-stimulating effects, either from beta-lactoglobulin or whey protein in its entirety."

Although whey may affect immune system function, it is unlikely the weight of this response will come from lactoferrin because it is representative of only about 1 per cent of product weight and is cost-prohibitive for wide-scale production.

Mushrooms
There is significant interest in the use of mushrooms and/or mushroom extracts as dietary supplements based on theories that they enhance immune function and promote health. The therapeutic effects of mushrooms, such as anticancer activity, suppression of autoimmune diseases and allergy, have been associated with their immunomodulating effects.20

To some extent, select mushrooms have been shown to have stimulatory action on immune responsiveness, particularly when studied in vitro. In particular, the ability of selective mushroom extracts to modulate the differentiation capacity of CD4 (+) T cells to mature into T(H)1 and/or T(H)2 subsets will be discussed. (These cells mediate immune responses by controlling factors such as the inflammatory response.)21

But despite their widespread use for potential health benefits, there is a surprising paucity of epidemiological and experimental studies that address the biologic activities and safety of mushrooms after oral administration to animals or humans.

However, researchers from The Hong Kong Polytechnic University investigated the toxicity of effects of four weeks Lingzhi (Ganoderma lucidum) supplementation on a range of liver and renal toxicity markers.22 Fasting blood and urine from 18 healthy adults were collected before and after four weeks supplementation with a commercially available encapsulated Lingzhi preparation (1.44g/day Lingzhi, equivalent to 13.2g/day fresh mushroom) or placebo. No significant change in any of the variables was found. Theses results showed no evidence of liver, renal or DNA toxicity with Lingzhi intake. This study shows that this form of mushroom use over the short term is safe.

Data from the same group investigated the in vitro antioxidant capacity of 10 days on a slightly lower dose of Lingzhi on biomarkers of antioxidant status, coronary heart disease (CHD) risk and DNA damage.23 In this double-blind, placebo-controlled, cross-over trial, blood and urine samples were collected from healthy volunteers at 0 (fasting) and 45, 90, 135 and 180 minutes post-ingestion of 1.1g Lingzhi. Repeat fasting samples were collected after 10 days' supplementation with 0.72g/day Lingzhi. The acute response (up to three hours) was also investigated with a larger dose (3.3g) of Lingzhi in a sub group of seven subjects.23

Results showed that the total antioxidant capacity (as the FRAP value) of an aqueous suspension of Lingzhi was 360micromol/g. Ingestion of Lingzhi caused a significant post-ingestion increase in plasma antioxidant capacity, with peak response at 90 minutes. Average increase of 29+/-11 per cent in urine antioxidant capacity was seen within three hours of ingestion.

After 10 days' supplementation with 0.72g/day Lingzhi, fasting plasma lipid standardised alpha-tocopherol concentration and urine antioxidant capacity increased. Fasting plasma ascorbic acid and total alpha-tocopherol concentrations and erythrocyte SOD and GPx activities increased slightly but nonsignificantly with supplementation. Plasma lipids and uric acid tended to decrease, but changes were not statistically significant. No deleterious effects on measured variables were seen.

Results indicate that Lingzhi intake causes an acute increase in plasma antioxidant capacity.23 The pattern of biomarker response after supplementation indicated possible benefit in terms of antioxidant status and CHD risk, but further study is needed to elucidate the nature and longer-term effects of the absorbable antioxidants from Lingzhi.

The future
Like immune-compromising disease states, the immune support and condition-specific prevention arenas are poised for explosive growth and innovative formulations. There are a many other active nutrients demonstrated in recent studies to act as potent immunomodulators based on current research, including vitamin E,24 nucleotides,²⁵ flavonoids26 and glutamine.27

However, some fundamental flaws should be considered when looking at all of these studies. One example of why caution must be taken can be seen with the use of whey extracts.28 It has been reported that the use of beta-lacatoglobulin preparations to stimulate immune cells in vitro can be caused by endotoxin contamination.28 Therefore, the immune responses seen with beta-lactoglobulin could be reported as a positive benefit of beta-lactoglobulin, when in reality it is because of supplement contamination as a result of poor GMPs.

Similar reports have shown the presence of bacterial and fungal contamination of many other bioactives.²⁹ This gives more weight to the importance of raw ingredient selection and screening for the general public, and researchers investigating the beneficial influences of functional foods/dietary supplements on immune system responses.

Mark J Tallon, PhD, is chief science officer of OxygeniX, a London-based consultancy firm specialising in claims substantiation, product development and technical writing.
www.oxygenix.com
Dr Tallon is also co-founder of Cr-Technologies, a raw-ingredients supplier.
www.cr-technologies.net

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