Possible roles of oxidative stress, local circulatory failure and nutrition factors in the pathogenesis of hypervirulent influenza: Implications for therapy and global emergency preparedness

Abstract

A pandemic with some highly virulent and also highly contagious form of avian influenza may be the most serious health threat facing the world today. Nutrients such as selenium and glutathione stimulate the antiviral immune response, counteract activation of the transcription factor NF-kappaB which is needed for replication of the influenza virus, will help to protect the mitochondria of infected organs, and counteract cytokine-induced activation of neutral sphingomyelinase, while the acid sphingomyelinase is inhibited by carnitine. A combination of high-dose coenzyme Q₁₀, acetylcarnitine and lipoic acid will also help to defend mitochondrial function, while a combination of selenium, glutathione (or some GSH precursor), antioxidants (which may include taurine and melatonin) and high-dose 5,6,7,8-tetrahydrobiopterin or 5-methyltetrahydrofolate (plus a stable prostacyclin analog?) may help to counteract local circulatory failure as a consequence of too much oxidative stress. It is imperative for the sake of better global contingency planning to test new treatment strategies (emphasizing antioxidant protection and defense against local circulatory failure) on human patients with malignant influenza, as well as on experimental animals suffering from the same disease, as soon as possible. It may also be very important to enhance global production and stockpiles of high-quality protein foods with a composition matching the needs of patients suffering from severe infectious diseases.

• Avian influenza
• pathogenesis
• oxidative stress
• nutrition
• selenium

Introduction

In discussions about emergency preparedness for a possible global pandemic of hypervirulent influenza, one factor is conspicuously absent not only when the matter is discussed in political fora, but also in most of the papers published in medical journals, i.e. the possible role of nutrition factors in host vulnerability to the disease. It has been known for a long time that morbidity and mortality from several other infections (including tuberculosis, diarrhoeal disease among small children, other lower respiratory infections and HIV disease) depend heavily on nutrition status, with protein-energy malnutrition and several micronutrient deficiencies all being important [1–3]. There is no reason to believe that influenza should be different in this regard compared to other (and better studied) infections. In particular, it must be expected that consequences of nutritional deficiency conditions that weaken the Th1-mediated immune response must be qualitatively similar in all viral infections including influenza, with a weakening of the Th1 response causing enhanced morbidity and – for the more dangerous infections – enhanced mortality.

Why protein intake is important

A normal immunological response depends on very fast replication of various types of leukocytes (or their progenitor cells) at the same time as amino acids are also needed for production of immunoglobulins, complement factors, and acute phase proteins. These processes depend on adequate supplies of several different amino acids, membrane lipid components, micronutrients and conditionally essential nutrients including nucleotides or nucleosides. But protein intake is also important because of the important role of the nonessential amino acid glutamine as fuel for various classes of leukocytes [4], [5]. Many (perhaps all) of the leukocytes have limited capacity to utilize glucose as fuel. Instead they need large quantities of glutamine for optimal function [4], [5]. Part of the glutamine used by leukocytes comes directly from the diet, but much of it is produced in skeletal muscle at the same time as other amino acids are degraded [5]. The capacity for glutamine production by skeletal muscle depends (in healthy or moderately ill persons) on the level of physical activity (because of enhanced consumption of amino acids as respiratory fuel when physical activity is enhanced) [6], but also on the availability of other amino acids (including leucine, isoleucine and valine) that can be used as fuel by the muscle cells. This will be partly controlled by the diet (composition and total daily food energy intake) and partly by the rate of degradation of muscle protein (in protein-catabolic conditions). The total mass of skeletal muscle must also be assumed to play an important role (with the total capacity for amino acid consumption as respiratory fuel and for glutamine production being enhanced in direct proportion to the total skeletal muscle mass). In famine situations it must therefore be expected that the capacity of glutamine synthesis by skeletal muscle will be reduced both as a consequence of wasting of skeletal muscle and because the intake of protein is too low. These two factors must be expected to interact with each other in a multiplicative fashion, which could be one of the main reasons for the high mortality from infectious diseases often observed in famine situations.

The growth rate of leukocytes is also affected by their glutathione status [7]. It may be possible that this can be largely explained by the important role played by GSH in the chain of electron transport from NADPH to ribonucleotide reductase. Ribonucleotide reductase can use either glutaredoxin or thioredoxin as reducing cofactors. Following oxidation, the reduced form of glutaredoxin is regenerated by reaction with GSH, while the reduced form of thioredoxin is regenerated by the enzyme thioredoxin reductase. Thioredoxin reductase is a selenoprotein and also a flavoprotein using NADPH as reducing cofactor [8]. Glutathione reductase, needed for regeneration of GSH from GSSG, is also a flavoprotein using NADPH as reducing cofactor. Riboflavin deficiency and niacin/tryptophan deficiency (causing NADPH depletion) may thus be expected to interfere with both of these alternate pathways for transport of electrons needed for reduction of ribonucleotides to deoxyribonucleotides. But one of these pathways also has a specific requirement for selenium, while the other has a specific requirement for sulfur amino acids needed for synthesis of glutathione.

Glutathione is also important for cellular antioxidative defense, i.e. as reducing cofactor for glutathione peroxidases and 1-Cys peroxiredoxin [9]. This is probably the main reason why it functions as an antiapoptotic factor in various cell types, including different types of leukocytes [10], [11]. Poor GSH status in the leukocytes may thus be expected to inhibit their growth or recruitment at the same time as their mortality rate will be enhanced.

Forms of malnutrition that specifically can weaken the antiviral immune response

Glutathione has also been found to affect the pattern of cytokine secretion in both experimental animals and humans, with good GSH status favoring the secretion of cytokines associated with the Th1 response while poor GSH status is attended by diminished secretion of Th1-associated cytokines [12–14]. Selenium status is also important because good selenium status is associated with enhanced expression of high-affinity and/or medium-affinity interleukin (IL)-2 receptors, i.a. on NK cells [15–17]. While good GSH status may help to enhance IL-2 production [14] (as one of the Th1-associated cytokines), good selenium status will help to make NK cells [16] and cytotoxic CD8⁺ T lymphocytes [17] more sensitive to its stimulating actions. The function of cytotoxic CD8⁺ cells has also been reported to depend on vitamin B₁₂ status, with B₁₂ deficiency causing impairment of CD8⁺ function [18]. NK cells and cytotoxic CD8⁺ T lymphocytes are important effector cells in the antiviral immune response.

It should be noted that not only is selenium intake very low in certain parts of Africa [19] and Asia [20], but the intake of sulfur amino acids is also much lower in large parts of sub-Saharan Africa than it is in Europe and North America [21]. This is in large measure a consequence of a combination of poverty and large cultivation (and consumption) of protein-poor staple foods such as cassava [22]. But sulfur deficiency in the soil (e.g. as a consequence of frequent anthropogenic fires on the savanna [23] attended by large losses of sulphur [24] and selenium in the form of SO₂ and SeO₂ in the smoke) may also be an important causative factor.

The antiviral immune response is attended by enhanced tryptophan degradation

The Th1 response is important not only for defense against viral pathogens, but also against intracellular bacteria including Mycobacterium tuberculosis. One of the effector mechanisms employed is to try to starve the pathogen of tryptophan, which happens through interferon-gamma (IFN-γ)-induced induction of the tryptophan-degrading enzyme indole-2,3-dioxygenase (IDO) [25]. This can help to limit the growth of cellular pathogens living in the leukocytes (but perhaps not tubercle bacilli because they grow too slowly) and may also help to limit the production of viral proteins.

But IDO induction is a double-edged sword because tryptophan deficiency can also limit leukocyte proliferation and thus be immunosuppressive. The latter effect must be expected to be more important when the dietary intake of tryptophan is marginal, either because the total protein intake is too low or because of large consumption of traditional maize varieties (with little lysine and tryptophan).

Role of NF-kappaB in control of influenza virus replication

Nutrition factors can also have an important effect on rates of pathogen replication. Pathogen growth can sometimes be inhibited by deficiency of some nutrient that is equally as important for the pathogen as it is for the host. But opposite effects can also be seen, as in the case of glutathione limiting the growth of tubercle bacilli [26]. The oxidatively activated transcription factor NF-kappaB stimulates the expression of the HIV-1 viral gene [27]. NF-kappaB activation is inhibited by GSH [27] and also by selenium-dependent antioxidative enzymes [28], which is probably one of the main reasons why the mortality rate in HIV patients is enhanced by poor selenium status. In an American cohort study of a group of HIV-positive drug addicts that were followed over a period of 3.5 years, mortality was enhanced by a factor of 19.9 in those patients who were defined as selenium-deficient, i.e. those who had less than 85 µg selenium per liter of blood plasma [29]. Mortality in vitamin B₁₂-deficient subjects was enhanced by a factor of 8.3 [29].

It has recently been reported that NF-kappaB also stimulates replication of the influenza virus, and that it is obligatory for replication of the virus [30], [31]. Even though no epidemiological data are available similar to those studies that have been carried out with HIV patients, it must be highly plausible that selenium and GSH status may have a significant impact on the behaviour of influenza viruses as well, with poor selenium or GSH status leading to enhanced NF-kappaB activation and more rapid replication of the virus, which again means enhanced virulence.

Role of reactive oxygen and nitrogen species in the pathogenesis of influenza

Oxidative stress is enhanced in all infectious diseases. There is more than one reason for this, an important factor being the use of reactive oxygen species (leukocyte respiratory burst), reactive halogen and pseudohalogen species (made by myeloperoxidase, eosinophil peroxidase, lactoperoxidase, and similar enzymes) and nitric oxide (NO) as weapons for killing cellular pathogens. But infectious diseases are also attended by enhanced mitochondrial production of reactive oxygen species (ROS). This is a consequence of enhanced secretion of cytokines such as tumor necrosis factor (TNF)-α that may cause activation of sphingomyelinases [32]. Sphingomyelin is then degraded to form sphingosine and ceramide. Ceramide itself or a ganglioside metabolite (or both) may subsequently enter the mitochondria where they will cause a partial block of electron flow through the respiratory chain. At the same time as this causes inhibition of mitochondrial ATP production, it also enhances the leakage of electrons from the respiratory chain before they reach the end of the chain and can be used for making water molecules plus ATP in the cytochrome oxidase reaction [32]. The ratio between rates of superoxide anion radical production and ATP production in the mitochondria will thus be enhanced.

The resulting intramitochondrial oxidative stress on the cell must depend not only on the rate of ROS production, but also on the mitochondrial capacity for scavenging of superoxide anion radical and H₂O₂. In nucleated cells catalase is found mainly in the peroxisomes, but not in mitochondria. Mitochondrial scavenging of H₂O₂ seems to depend mainly on glutathione peroxidase-1 (Gpx-1) [33] and peroxiredoxins [34]. The level of the selenoprotein Gpx-1 depends heavily on selenium intake. The enzyme follows tert-uni ping pong kinetics [35], which means that the rate of H₂O₂ scavenging (at a given concentration of the oxidizing substrate) is determined by the product of the concentrations of the enzyme itself and the reducing cofactor GSH. But intracellular and intramitochondrial GSH concentrations are reduced when the intake of sulfur amino acids (or any of those B-group vitamins that are needed for normal GSSG reduction) is too low.

It has also been reported that the activation of neutral sphingomyelinase is inhibited by GSH and by glutathione peroxidase [36], [37], while the activation of acid sphingomyelinase is inhibited by carnitine [38]. Poor GSH or selenium status may thus have a double deleterious effect when sensitizing TNF-α target cells to the pro-oxidant effects of this cytokine. But the production of TNF-α is also under partial control of nutrient factors, since the expression of the TNF-α gene is under control by various oxidatively activated transcription factors including NF-kappaB [39]. Poor antioxidant nutrient status (e.g. GSH depletion) may thus lead to enhancement of TNF-α production at the same time as it also sensitizes various target cells to its pro-oxidant action [40].

It has been reported that peroxynitrite plays an important role in the pathogenesis of ordinary influenza [41]. This can probably be explained as due to a combination of enhanced mitochondrial production of superoxide anion radical for reasons just explained, and enhanced expression of leukocyte (inducible) NO synthase (NOS-2). Superoxide anion radical and NO react with each other in a very fast (diffusion-controlled) reaction to produce peroxynitrite.

GSH can also react with NO, making S-nitrosoglutathione, which may be considered a less reactive storage or transport form of NO, but which is also a powerful antioxidant [42]. This reaction is reversible (so that NO can be regenerated from S-nitrosoglutathione), but S-nitrosoglutathione can also be degraded in reactions forming other reaction products instead of NO. This happens when S-nitrosoglutathione reacts with superoxide anion radical (forming nitrite and nitrate) [43] and also when it is degraded by the enzyme alcohol dehydrogenase-3 (forming glutathione sulfinamide) [44]. It may thus be expected that good GSH status will help to limit the production of peroxynitrite, while GSH depletion will have an opposite effect.

It has also been reported that peroxynitrite can be scavenged by the selenoproteins Gpx-1 and selenoprotein P (in extracellular compartments) [45]. But because of the ping pong kinetics of the enzyme [35] the effect of Gpx-1 depends not only on selenium status but also on intracellular GSH concentrations. Selenoprotein P can use different reducing cofactors, but thioredoxin is probably the most important one [46].

Oxidative stress-induced impairment of the local circulation

In the healthy organism, vascular resistance is bidirectionally regulated; it thus depends on the balance between several different vasoconstrictor and vasodilator signal substances. Among the latter, two have been especially well studied, i.e. NO (which is produced by endothelial NO synthase) and prostacyclin (PGI₂ and PGI₃), which is produced by endothelial prostacyclin synthetase.

Prostacyclin synthetase, however, can easily be inactivated as a consequence of enhanced oxidative stress. The enzyme is inactivated by peroxynitrite [47], by very low concentrations of some of the fatty acid hydroperoxides [48] and also by oxidized serum lipoproteins [49]. But the production of NO is also vulnerable to enhanced oxidative stress, especially when the production of superoxide anion radical is enhanced, e.g. by activation of endothelial NAD(P)H oxidase [50]. This will enhance the production of peroxynitrite at the same time as less NO will reach the vascular smooth muscle cells [50]. When oxidative stress in the endothelium is enhanced, this also causes enhanced oxidative degradation of 5,6,7,8-tetrahydrobiopterin [51]. This substance is one of the cofactors needed by NO synthase, and deficiency of this cofactor causes the enzyme to make superoxide anion radical instead of NO [51]. This problem can, however, be corrected by supplementation with high doses of 5,6,7,8-tetrahydrobiopterin [52] or 5-methyltetrahydrofolate [53]. The latter substance can substitute for some of the 5,6,7,8-tetrahydrobiopterin needed as cofactor for NO synthase causing normalization of the function of the enzyme (when partially unsaturated with the normal cofactor) [54], but must then be administered at a dosage level one order of magnitude higher than needed for saturation of ordinary folate-dependent enzymes (at a dosage level of 5 mg/day for adult patients with cardiovascular diseases, but it may be possible that even more may be needed when the oxidative stress is very high because of severe infection).

Can a combination of mitochondrial inhibition and local circulatory failure explain irreversible organ failure in severe influenza and other hypervirulent infections?

When two of the most important vasodilators are simultaneously taken out, more or less, as a consequence of excessive oxidative stress, it is not difficult to see that this might have potentially severe consequences for maintenance of normal perfusion of the affected organ. But reactive oxygen and nitrogen species can also inhibit a number of mitochondrial enzymes, thus inhibiting mitochondrial ATP production. Aconitase is very sensitive to inhibition by superoxide anion radical [55]. α-Ketoglutarate dehydrogenase is inhibited by H₂O₂ [56]. Cytochrome c oxidase is inhibited by NO [57], and peroxynitrite damages complexes I and II of the electron transport chain [57], ATPase [57], aconitase [57], and Mn-superoxide dismutase [57]. NO functions as a reversible inhibitor of cytochrome c oxidase, but competes with molecular oxygen for binding to the enzyme [58]. When functioning as a competitive inhibitor of the reaction with oxygen, it enhances the oxygen partial pressure needed for maximal activity of the enzyme, while hypoxia will enhance the sensitivity of the enzyme to the toxic effects of high levels of NO.

Oxidative stress can, moreover, also enhance degradation of coenzyme Q₁₀ [59] at the same time as too much oxidative stress can inhibit the synthesis [60], [61] of this vitally important electron shuttle (and antioxidant [59]). Coenzyme Q₁₀ is degraded by oxidative stress much faster than α-tocopherol, β-carotene, and lycopene [59]. The rate of coenzyme Q₁₀ synthesis depends on the availability of methionine as precursor for making S-adenosylmethionine (SAMe) [61], but too much oxidative stress oxidizes methionine to methionine sulfoxide, thus making it unavailable for SAMe biosynthesis [60]. It must be expected that there must be a synergistic interaction here between oxidative stress (because of infection) and a low dietary intake of methionine. There are two different stereoisomers of methionine sulfoxide, which can be reduced back to methionine by two different methionine sulfoxide reductases (methionine sulfoxide reductase A and methionine sulfoxide reductase B), both using thioredoxin as their reducing cofactor [62]. One of these (MsrB) is itself a selenoprotein in many tissues [63], and both of them are indirectly dependent on the selenoprotein (and flavoprotein) thioredoxin reductase, being needed for regeneration of reduced thioredoxin.

The ratio of SAMe to S-adenosylhomocysteine (that functions as a competitive inhibitor of transmethylation reactions [64]) can also be reduced as a consequence of folate, vitamin B₁₂ or vitamin B₆ deficiencies. But folate is easily degraded by oxidative stress. A combination of ROS and iron can cleave its pteridin ring [65], and depletion of this vitamin is commonly observed both in smokers [66] and among alcoholics [65]. Diet-induced deficiency of folate and/or vitamin B₁₂ is common in many poor countries, with the situation as regards vitamin B₁₂ being especially alarming in parts of India (where as much as about 75% of the subjects in a hospital population comprising patients as well as healthy persons were found to have metabolic signs of cobalamin deficiency) [67].

One must expect that coenzyme Q₁₀ depletion and cytochrome c oxidase inhibition (by NO or peroxynitrite) will synergize with ceramide (because of cytokine-induced activation of sphingomyelinases) [32] as causes of inhibition of electron flow through the respiratory chain, which will in turn cause enhanced leakage of electrons and enhance the rate of superoxide anion radical production [32]. Much of this leakage comes from autoxidation of redox-active groups in complex I (most probably the Fe-S cluster N2) [68], and any factor damming up electrons in complex I will lead to enhancement of the rate of its reaction with molecular oxygen. The rate of this reaction (causing production of superoxide anion radical) must, moreover, also be expected to depend on the intramitochondrial O₂ partial pressure. But enhanced production of ROS in the mitochondria must be expected to lead to enhancement of the rate of oxidative degradation of coenzyme Q₁₀, opening the possibility of a highly vicious circle (with depletion of coenzyme Q₁₀ enhancing the rate of mitochondrial ROS production at the same time as too much of the latter may lead to exacerbation of the coenzyme Q₁₀ depletion). It is not difficult to see that this is a very dangerous mechanism which in principle could turn fatal.

It may be noted that high age per se causes enhancement of the rate of superoxide anion radical in the mitochondria (because of the age-related accumulation of mitochondrial mutations leading to depletion of normal mitochondrial genes causing decreased production of mitochondrially encoded proteins) [68]. High age must therefore be expected to synergize with the other factors already mentioned as causes of enhanced mitochondrial production of superoxide anion radical – which might possibly be one of the main reasons why mortality from ordinary influenza (and also several other infections) is enhanced in geriatric patients. It is not unreasonable to assume that the synthesis of mitochondrially encoded proteins may be further impaired by protein-energy malnutrition – thus making high age, protein-energy malnutrition and infectious disease (causing ceramide-induced impairment of mitochondrial electron flow) a very deadly combination.

It must be expected that impaired mitochondrial function (because of a combination of coenzyme Q₁₀ depletion and mitochondrial enzyme inhibition) and impaired local circulation (because of the combination of excessive production of superoxide limiting the availability of NO as a vasodilator and reduced synthesis of prostacyclin) must interact synergistically with each other as causes of reduced mitochondrial ATP production at the same time as local hypoperfusion also will enhance the mitochondrial toxicity of NO (being produced by activated leukocytes). It is not difficult to see that this combination of circumstances may offer a highly plausible explanation for tissue necrosis and organ failure not only in hypervirulent influenza, but quite possibly also in other severe infections, perhaps including cerebral malaria and fatal meningococcal disease. Something similar may very likely happen in various cases of fatal poisoning with substances that function as redox-cycling agents making large quantities of superoxide anion radical, e.g. paraquat and orellanine.

Antioxidative protection in lung inflammation

Ordinary influenza is primarily a respiratory infection, and this was also the case with the Spanish flu. It should be noted that glutathione-, selenium-, and riboflavin- and NADPH-dependent enzyme systems are not only important for scavenging H₂O₂ and organic hydroperoxides, but also for repair of oxidatively damaged proteins. Thioredoxin and thioltransferase (glutaredoxin) help to repair abnormal disulfide bonds (such as protein-protein disulfides and glutathionyl-protein disulfides) [69] and depend, respectively, on the NADPH-dependent selenoprotein (and flavoprotein) thioredoxin reductase and on GSH for regeneration of their reduced forms. Repair of oxidatively damaged methionyl groups in proteins is carried out by methionine sulfoxide reductases that use thioredoxin as their reducing cofactor [62], while one of the methionine sulfoxide reductases is itself a selenoprotein [63].

GSH also has a protective function more specific for the lungs, since the alveolar epithelial lining fluid has been found to contain about 100 times more GSH (in normal persons) than is found in the extracellular fluids of most other tissues [70].

On this background, it may be reasonable to expect that poor selenium or GSH status may contribute to exacerbation of any kind of lung infection, with the combination of selenium and GSH depletion being especially dangerous. It is possible that this could be one of the main reasons for high mortality rates from lower respiratory infection among small children in developing countries and especially in famine situations, as exemplified by the situation in Somalia in 1992 [71].

The 1918 Spanish influenza pandemic virus has now been reconstructed, which makes it possible to understand its exceptional virulence [72]. During experimental infection in mice, the animals were found to have developed necrotizing bronchitis and bronchiolitis and moderate to severe alveolitis on day 4 post infection [72]. The alveolitis was composed of neutrophils and macrophages [72]. These cell types may thus play a key role in the pathogenesis of malignant influenza. It has been shown, however, that enhancement of the mitochondrial production of ROS in endothelial cells is associated with enhanced expression of various adhesion factors that must be expected to enhance the accumulation of neutrophils in the infected lung tissue [73]. This problem will presumably be exacerbated by malnutrition causing impairment of the antioxidative defense system of the endothelial cells, while saturation of selenium- and GSH-dependent antioxidative enzyme systems in the endothelium must be expected to be protective. It has been demonstrated that experimental selenium deficiency is associated with increased neutrophil adherence and adhesion molecule mRNA expression in endothelial cells [74], while glutathione peroxidase mimics prevent TNF-α- and neutrophil-induced endothelial alterations [75].

Expression of the chemokine IL-8 is positively regulated by NF-kappaB [76], while NF-kappaB activation is inhibited by GSH [27] and selenium-dependent antioxidative enzymes [28]. It may thus be expected that neutrophil infiltration in the lungs of influenza patients may be enhanced by selenium or glutathione depletion not only because of enhanced expression of endothelial adhesion factors, but also because of enhanced production of an important chemoattractant.

It has, moreover, also been reported that selenium and vitamin E participate in the regulation of the complement-neutrophil-reactive oxygen activation feedback mechanism-mediated inflammatory response, with selenium and vitamin E supplementation causing inhibition of the complement-neutrophil-reactive oxygen activation feedback mechanism with reduced ROS production and complement activation in mouse in vivo vasculitis models of skin, lung, and liver [77], [78]. It has also been reported that sodium selenite has a marked inhibitory effect on the hemolysis induced by complement fixation in vitro, as well as on mouse complement activation in vivo induced by endotoxin, inulin or aggregated IgG [79]. This might perhaps help to explain the strong protective effect of sodium selenite (with strong mortality reduction) that has been observed in Chinese patients suffering from epidemic hemorrhagic fever – an infectious disease which is otherwise very often lethal [80]. Selenium-dependent enzymes have also been reported to be very important for prevention of intestinal inflammation [81].

Therapeutic intervention possibilities

It may be concluded that not only are nutritional factors very important for the function of the immune system, but they may also be critically important for the protection of host tissues against the excessive oxidative and nitrosative stress probably attending malignant influenza, at the same time as it is also possible that they may have an important effect (via NF-kappaB) on the rate of influenza virus replication. It can also be seen that a number of positive feedback regulatory cycles (or vicious circles) may play an important role in the pathogenesis of malignant forms of influenza and that good nutrition status may serve to dampen these vicious circles (e.g. the probable self-stimulation of virus replication mediated by oxidative stressors, cytokines and NF-kappaB), while they may be strengthened by various forms of malnutrition, thus enhancing the likelihood of a fatal outcome.

There is therefore strong reason to expect that morbidity and mortality will be higher for any form of influenza in poorly nourished populations, with intakes of selenium and sulfur amino acids being perhaps the most critical factors, but also B-group vitamins needed for the normal operation of the glutathione reductase and thioredoxin reductase systems (i.e. riboflavin, niacin/tryptophan and thiamin), as well as several other micronutrients and conditionally essential nutrients needed for optimal function of the immune system. In case of a global pandemic with some highly virulent form of influenza, there is reason to expect that mortality rates will be much higher in most of sub-Saharan Africa than in North America, but it is also possible that mortality rates will be substantially higher in many of the countries of Western and Central Europe than in the United States and Canada because of lower selenium intakes in many parts of Europe [82], [83].

But there is another side to this coin. When nutrition factors are important for so many different aspects of the disease process, it also means that there should be good possibilities of therapeutic intervention with a quadruple aim: (i) optimizing antioxidant protection of the lungs and other organs that might be attacked by the virus; (ii) defending mitochondrial function and the local circulation in these organs; (iii) reducing oxidative activation of NF-kappaB in an effort to minimize the stimulating effect of NF-kappaB on the replication of the virus; (iv) optimizing the function of the immune system, with special emphasis on the function of NK cells and cytotoxic CD8⁺ cells.

For optimizing antioxidant protection and mitochondrial function, a multifactorial intervention strategy may be warranted, using several different substances that have different properties and different mechanisms of action, so that they may interact synergistically with each other. While the importance of sulfur amino acids/glutathione and selenium is obvious from what has been explained above, other substances should probably also be used to obtain a maximal protective effect, including high-dose coenzyme Q₁₀ (90 mg/day or more), taurine (500 mg/day or more), melatonin, lipoic acid, and acetyl-carnitine.

Coenzyme Q₁₀ should be given not only because of its antioxidant role, but first of all in an effort to defend mitochondrial ATP production and avert the potentially fatal vicious circle of progressive depletion of coenzyme Q₁₀ and enhancement of mitochondrial ROS production. Taurine functions as an important intracellular antioxidant with antimutagenic effect [84], [85], but the mechanism for its antioxidant action has not been well understood. There is reason to expect, however, that it may help to diminish the pro-oxidant catalytic effect of phosphate-bound iron [86] through formation of mixed complexes where the iron atom is attached to a phosphate group on one side and the sulfonic acid group of taurine on the other – with the three oxygen atoms from the phosphate group and the three oxygen atoms from the sulfonic acid group forming an octahedron, which is the preferred coordination with oxygen both for ferrous and ferric iron [87]. Taurine has been reported to protect endothelial cells against damage induced by high doses of IL-2 [88], and presumably may also help to protect them against other cytokines (or other signal substances) functioning as oxidant stressors.

As with coenzyme Q₁₀, lipoic acid also has a double role both as an antioxidant [89] and as a coenzyme necessary for the function of various mitochondrial enzymes. Combined supplementation with lipoic acid and acetyl-carnitine has been reported to partly restore mitochondrial function in old experimental animals [90]. During severe infectious disease, carnitine supplementation may also be beneficial because it will help to counteract cytokine-induced activation of acid sphingomyelinase [38]. Melatonin cannot be considered a nutrient, but has properties making it potentially highly useful in acute medicine whenever oxidative stress is strongly enhanced. It is not only itself an excellent antioxidant [91], but acting as a hormone, it also enhances the expression of a number of antioxidant enzymes [92] and ameliorates mitochondrial dysfunction [93]. Melatonin also has immunomodulatory effects [94] that may be useful not only in cancer [95], but also in connection with viral infections [96], [97].

For defending the local circulation in severely affected organs, it seems logical to use a combination of either 5,6,7,8-tetrahydrobiopterin or high-dose 5-methyltetrahydrofolate (5 mg/day or more) with a stable prostacyclin analog.

Necessity of improved emergency preparedness

Even a cursory glance at the food supply situation in several countries in Asia, sub-Saharan Africa and Latin America is enough to show that the world is extremely poorly prepared to tackle a new global pandemic with an avian influenza equally as virulent as the 1918 Spanish flu virus or perhaps even worse. Unless the global production capacity for influenza vaccines and drugs is vastly expanded, it must be expected that neither vaccines nor drugs will be available for more than a small proportion of the world's population and also that a majority of those who will receive vaccine (if available) or drugs will live in the affluent countries [98]. But it must be expected that the disease will strike much harder among the world's poor populations (because of much poorer diet) than among the rich ones.

For improving the global emergency preparedness, step one should be to start – as soon as possible – doing experiments with antioxidative therapies for influenza both in animal models (using some of the highly virulent strains of influenza virus now available) and in human cases of hypervirulent avian influenza. If successful, the results of such trials should be employed to formulate guidelines for treatment of the disease not only for the benefit of clinicians handling cases of hypervirulent influenza, but also as a basis for emergency preparedness policy planning at both the international and national level.

The world has considerable stocks of cereals and other energy-rich staple foods (e.g. sugar, various dietary fats and oils), but very limited stocks of dried protein-rich foods with a composition matching the needs of patients suffering from severe infectious diseases. For substantial reduction of the number of deaths associated with a pandemic of some highly virulent form of influenza, it will be imperative both to enhance the production capacity of high quality protein foods (especially in the poor countries of Asia, Africa and the Caribbean) and to enhance stockpiles of dried and not too expensive, but high-quality protein foods (e.g. skimmed milk powder, whey powder, fish protein concentrate type B). Most likely it will also be necessary to enhance global production capacity and stockpiling for several of those other substances that have been mentioned above. For populations now selenium-deficient compared to what may be needed for optimal protection against dangerous viral diseases [2] (e.g. in most of Europe) it may also be important to enhance – as soon as possible – selenium intake through the ordinary diet.