More about hypervirulent avian influenza: Is the world now better prepared?

Abstract

The World Health Organization (WHO) no longer regards the Spanish flu pandemic as a worst case scenario as the basis for contingency planning, as to how to meet a possible pandemic with hypervirulent H5N1 influenza. The worldwide fatality rate among confirmed cases has increased from 43% in 2005 to 69% in 2006, while in Indonesia it was 82% in 2006. What the world now needs to be prepared for is a pandemic with something that is equally as transmissible as ordinary influenza virus, but which may have a case fatality rate of 80% or more, if not treated with therapeutic methods much better than those available today (i.e. treatment methods that have not been sufficient to hinder the average case fatality rate from climbing to as much as 82% in Indonesia in 2006). Not only are current treatment methods not efficacious enough, but the situation is also very far from satisfactory as regards vaccine development and production – which means that the world must still be considered to be almost totally unprepared, if a pandemic with hypervirulent H5N1 influenza should start tomorrow. A very detailed description of new experimental work is given to make it possible to understand better some of the main elements of the attack strategy used by the enemy – so that this ‘military intelligence’ information can be used as the basis for a hopefully more rational strategy of defense. The enemy neutralizes a system used for very early detection of invasion with RNA viruses, leading to total or partial immobilization of associated early response defensive weapon systems. The proposed defense strategy comprises two elements: 1) keeping the patient alive until rescue forces (i.e. a good adaptive immune response) can arrive, and 2) making it possible for the rescue forces to arrive as soon as possible. Some tactical principles and weapons that may be used for defense purposes are also proposed, partially on the basis of animal experiments with both lethal influenza and other highly lethal viruses. A main challenge is to reduce pulmonary inflammation causing alveolar edema without simultaneously hindering the development of a good adaptive immune response. Several practical suggestions are given as to how this possibly might be done. However, it is imperative that the suggested new defense strategy (or therapeutic strategy) should be tested as soon as possible both in experimentally infected animals and in spontaneously occurring human cases of hypervirulent H5N1 influenza.

Introduction

Writing review or commentary articles in a highly dynamic field of research is associated with the risk that a manuscript that was reasonably well updated when written may have become wholly or partly outdated by the time it appears in print. This has been the fate of our article ‘Why is the world so poorly prepared for a pandemic of hypervirulent avian influenza?’ [1]. The main reason for this is the appearance of a new World Health Organization (WHO) report, following a meeting of a working group on 21–22 September 2006 [2]. The WHO has now officially and in very clear words stated that the risk scenario associated with a possible outbreak of pandemic H5N1 influenza should be considered far more serious than the assumption that was taken as a basis of emergency preparedness planning in most countries before this meeting – viz. that the mortality associated with the Spanish flu pandemic could be used as a worst case scenario for contingency planning [1]. The working group concluded that there is no reason to believe that hypervirulent H5N1 strains would become any milder if one of them should evolve to become a pandemic strain. Or, to use the words of the report:

‘One especially important question that was discussed is whether the H5N1 virus is likely to retain its present high lethality should it acquire an ability to spread easily from person to person, and thus start a pandemic. Should the virus improve its transmissibility by acquiring, through a reassortment event, internal human genes, then the lethality of the virus would most likely be reduced. However, should the virus improve its transmissibility through adaptation as a wholly avian virus, then the present high lethality could be maintained during a pandemic.’

What are the implications of WHO's new risk assessment?

What are the implications of the words just quoted – what does this mean? On its home page WHO gives a frequently updated statistical overview of all confirmed human H5N1 cases, by country and year, since the resurgence of H5N1 transmission to humans started in 2003. There were altogether 4 confirmed cases and 4 dead in 2003, 46 cases and 32 dead in 2004, 97 cases and 42 dead in 2005, and 116 cases and 80 dead in 2006. This means that the total number of confirmed human cases has increased each year from 2003 to 2006, giving no indication that the current epizootic (with scattered human spillover) of hypervirulent H5N1 influenza has culminated or that the situation worldwide has become stabilized in a way meaning that the acute danger is over. The fatality among confirmed human cases can be seen to have been reduced from 2003 to 2005, being 100% in 2003 (but with only n=4), 70% in 2004 and 43% in 2005. It might be speculated that this reduction of fatality is not entirely fortuitous, but might have happened as a consequence of earlier diagnosis and better treatment (because of more global attention to this disease and more clinical experience with the attempted therapy methods). But then the fatality again rose from 43% in 2005 to 69% in 2006 (all countries). There is no reason to believe, however, that this happened as consequence of a deterioration of treatment methods or slower diagnosis, comparing the situation in 2006 with that in 2005. A more worrying possible explanation is that it could have happened because of evolutionary change in the virus itself, with some of the H5N1 subclades now being even more virulent than was the case in 2005 (or that one subclade that was especially virulent even in 2005 may have become relatively more common as a cause of human outbreaks than it was before).

Indonesia was the country that had the largest number of confirmed H5N1 cases in 2006, with 56 confirmed cases and 46 dead (but 46 human cases and 37 dead at the time when the WHO report was written – the end of September) [2]. This gives an average lethality of 82% for Indonesia in 2006. Most cases were under the age of 30 years [2]. For unknown reasons, fatality was higher in females [2]. One-third of the cases registered until the end of September 2006 belonged to clusters [2].

Since Indonesia had the highest number, among all countries, of confirmed human H5N1 cases in 2006, and since there is also evidence from Indonesia of more human-to-human transmission than before (with many cases belonging to clusters), Indonesia may for the moment be considered that single country in the world where the probability of outbreak of a pandemic with hypervirulent H5N1 influenza should be considered to be highest, if this happens without reassortment with other influenza virus strains. (Although in principle it could happen almost anywhere, even in North or South America following dispersal of hypervirulent virus strains with migrating birds either from Northeast Asia or from Western Europe.) But the assumption of no change in lethality following a pandemic outbreak in Indonesia means that we are dealing with a disease capable of killing close to 80% of the population in countries that are at a similar economic level to Indonesia, and where the average diet is similar. This is comparable to or worse than the more recent historical estimates for the death toll during the Black Death in Europe – which may have killed about 60% of the population in Europe at that time [3]. Judging from the experience from the Spanish flu pandemic, when mortality was much higher in poor countries than in Western Europe and North America [4], [5], it may be possible that average death rates during a hypervirulent H5N1 pandemic would be considerably lower in Western Europe, North America, and Japan than in several African and Asian countries. But in much of sub-Saharan Africa, it seems possible that the average death rate could become even higher as a consequence, for instance, of low dietary intake of sulfur amino acids or poor selenium status [6], both of which are extremely important for antioxidant defense and for control of the activity of the transcription factor NF-κB, which has been reported to be required for replication of the influenza virus [6].

Other nutritional deficiency conditions, such as iron deficiency, zinc deficiency, lysine deficiency, tryptophan deficiency (especially when tryptophan-poor maize is a dominant local staple), vitamin A deficiency, and various B group vitamin deficiencies are commonly associated with poverty at the same time as they are geographically very widespread. In many cases they are not so specifically associated with particular geographical regions as may be the case with selenium deficiency and deficiency of sulfur amino acids in the average diet of the population in parts of sub-Saharan Africa [6]. Most of the nutrients concerned are needed for all growth processes at a cellular level, for instance iron as an obligatory cofactor for the enzyme ribonucleotide reductase (which means that it is required for DNA synthesis and repair). But a normal immunological response is dependent on very rapid proliferation of several types of leukocytes or their progenitor cells. It is therefore easy to understand why deficiency of almost any essential or conditionally essential nutrient needed for normal cellular growth may have a negative impact on the capacity of an organism to defend itself against dangerous bacteria or viruses. But it may be possible that this could be even more important in connection with infections caused by RNA viruses than for many other infections owing to the small genome size and therefore phenomenal rates of replication that are possible for RNA viruses, compared with the much slower replication observed for human cells. It is also possible that deficiency of nutrients needed as cofactors for enzymes specifically needed for the synthesis of DNA, but not for synthesis of RNA, may be especially harmful in connection with infections caused by RNA viruses (such as influenza, rotavirus infection, and measles), since they might be expected to affect the rate of mammalian cell replication more strongly than they affect the rate of RNA virus replication [7]. It may be possible that deficiency of all nutrients that directly or indirectly are needed for the function of ribonucleotide reductase could have this effect (i.e. nutrients such as iron, sulfur amino acids, selenium, riboflavin, niacin/tryptophan, and thiamine), and also nutrients needed for normal methylation of deoxyuridylate to form deoxythymidylate (such as folate, vitamin B₁₂, and methionine), while zinc is required as cofactor for thymidine kinase and hence for synthesis of deoxythymidylate by the salvage pathway. It is reasonable to speculate that some of the nutritional deficiencies concerned may interact with each other in a synergistic fashion as factors limiting the rate of DNA synthesis in leukocytes, e.g. if the activity of ribonucleotide reductase was reduced because of iron deficiency at the same time as the rate of electron transport to the reducing cofactors glutaredoxin and thioredoxin is impaired because of deficiency of sulfur amino acids (because glutathione is used as reducing cofactor for glutaredoxin), selenium (thioredoxin reductase being a selenoprotein) or riboflavin (thioredoxin reductase and glutathione reductase being flavoproteins). Vitamin A may be needed, as will be discussed later, for normal expression of a molecule called RIG-I that is used for early detection of viral RNA from several of the RNA viruses, including influenza virus.

The world population is much bigger now, however, compared with the situation during the 14th century before the Black Death started. It means that the total number of deaths would be expected to become much larger now, if we were to have a pandemic with an average mortality (relative to total population) comparable to the Black Death in the non-affluent countries of Africa and Asia. In terms of total number of deaths, it would be the worst catastrophe ever during the history of our species.

Is it possible that WHO's new risk assessment still might prove over-optimistic?

It may be possible, though, that even this very gloomy scenario could turn out to be over-optimistic. The Spanish flu pandemic ran in four different waves during the period 1918–20, with the first wave probably affecting the largest total number of previously non-immune people, but the total number of deaths was higher during the second than during the first wave [7], [8]. During the second wave, it is probable that the population of non-immune and therefore susceptible people was much smaller than it had been at the start of the first wave [7]. The lethality (among infected cases) must therefore most likely have been much larger during the second than during the first wave. There are two alternative theoretical explanations for how a second wave could come so soon after the first one. It might have happened because of antigenic drift (so that those who had been infected during the first wave could be infected once more), or because of one or more mutations causing enhanced transmissibility of the virus (in which case the second wave could have spread as a new pandemic among those who had been left non-immune after the first wave) [7].

Without RNA sequence data for viruses from both the first and second wave, it is not possible to prove or disprove either of these alternative hypotheses. But the very short time between the end of the first wave and the start of the next one (maybe they even overlapped in time) is an argument in favor of the second hypothesis. The same may also be said about the much higher lethality observed during the second wave (which cannot be explained only as a consequence of antigenic drift – in which case it is more plausible that mortality would have been smaller during the second wave than it was during the first one).

An American group has used hospital notification data during the 1918 influenza pandemic in the Canton of Geneva, Switzerland and epidemic modelling to estimate the reproductive numbers (i.e. average number of other persons infected by one infected person) of the first and second waves of the Spanish flu pandemic [9], [10]. These numbers were found to be R₁=1.49 (95% CI: 1.45–1.53) for the first wave and R₂=3.75 (95% CI: 3.57–3.93) for the second wave [9], [10]. In addition, the clinical reporting of influenza cases was estimated to be 59.7% (95% CI: 55.7–63.7) for the first wave and 83% (95% CI: 79–87) for the second wave [10]. It was surmised that the lower reporting in the first wave can be explained by a lack of initial awareness of the epidemic and the relative higher severity of the symptoms experienced during the fall wave [10]. The effect of hypothetical interventions was also modeled; it was found that effective isolation measures in hospital clinics at best would only ensure control with probability 0.87, while reducing the transmission rate by >76.5% guarantees stopping an epidemic [10]. The reproductive number of the second (autumn) wave has also been studied with four different methods in San Francisco, California [11]. Whereas the values of R obtained using the first three methods based on the initial growth phase were estimated to be 2.98 (95% CI: 2.73, 3.25), 2.38 (CI: 2.16, 2.60), and 2.20 (CI: 1.55, 2.84), the third method with the entire epidemic curve yielded a value of 3.53 (CI: 3.45, 3.62) [11].

More than doubling of the reproductive number from the first to the second wave of the Spanish flu pandemic in the Canton of Geneva in spite of probable strong reduction of the population density of infectible (i.e. non-immune) persons [7] must be taken as strong indirect evidence that the virus had indeed changed its transmissibility properties when the second wave of the Spanish flu pandemic started, and that it had become much more infective during the second wave than had been the case during the first wave. If it is assumed that the infectible population density was about halved from the first to the second wave, it can be loosely estimated that the infectivity of the virus could have increased by about a factor of 4 to 5 from the first to the second wave. It is far from obvious that this is something that could have happened as a consequence of antigenic drift alone, even though the possibility cannot be excluded (for reasons that will be explained below) that a case of antigenic drift associated with enhancement of the number of carbohydrate groups on the influenza virus hemagglutinin molecule might be associated with substantial enhancement of the rate of virus uptake in the cells, which might in turn lead to simultaneous enhancement of both the transmissibility and virulence properties of the virus.

The influenza virus hemagglutinin binds to receptor molecules on the cell surface which are glycoproteins that contain negatively charged carbohydrate groups. Electrostatic attraction may thus play an important role in the binding of viruses to the receptor molecules on the cell surface. But there are also many other negatively charged molecules both on the cell surface and in the intercellular space (including body fluids such as blood plasma or mucus secreted in various organs of the respiratory tract), and there is good reason to suspect that there may be much unspecific binding of viruses as a consequence of electrostatic forces to negatively charged molecules other than those receptor molecules they must bind to before they can be taken up by the cells. There will then be a competition between ‘productive’ binding of the virus particles to their specific receptors (which is required before proliferation of the virus in a cell can start) and ‘non-productive’ binding of the virus particles to all other kinds of negatively charged molecules either on the cell surfaces or in the intercellular space. The kinetics of virus uptake in the cells will then be determined not only by the stability constant of the virus–receptor complex, but also by the concentrations of all molecules forming ‘non-productive’ complexes with the virus as well as by the stability constants for all these ‘non-productive’ complexes.

Improvement of virus binding to its specific receptor can then happen in two different ways, either because of enhancement of the stability constant for the ‘productive’ complex with the specific viral receptor or because of a reduction of the stability constants for important ‘non-productive’ complexes with other negatively charged molecules that compete with the specific receptor for binding of the virus particles. But the viral hemagglutinin can itself bind oligosaccharides (through N-glycosylation) at positions other than the site binding with the specific viral receptor. These oligosaccharide groups are sulphated [12], [13], which means they will become negatively charged. It may be possible that introduction of new glycosylated sites on the hemagglutinin molecule and thus masking the peptide sequence below is one of the most important mechanisms used by the virus to avoid specific immunity because of the previous contact of the immune system with some of its very close relatives during earlier epidemics, i.e. this may be one of the most important mechanisms of immune evasion associated with the so-called antigenic drift of influenza viruses. But it is also possible that it may help to decrease the binding constants for ‘non-productive’ binding of the virus to negatively charged molecules other than the specific viral receptor(s). This might conceivably happen mainly as a consequence of enhancement of the total negative charge on the virus particle, causing reduction of the strength of non-specific electrostatic attraction to other negatively charged molecules, especially when considering binding to such other molecules that unlike several mucins [14] don't contain sialic acid. But the local geometry and electrical charge distribution of the part of the hemagglutinin molecule participating more directly in the binding with the viral receptor will presumably not be changed, so it may be possible that the strength of binding to the specific viral receptor is less affected than the more non-specific binding of the virus to several other negatively charged molecules.

It may thus be possible that higher transmissibility and higher lethality of the virus during the second wave of the Spanish flu pandemic may have had a common cause, i.e. one or more mutations that either enhanced the stability constant for the complex between virus hemagglutinin and the hemagglutinin receptor found both in the upper and lower human airways [7] or reduced the strength of ‘non-productive’ binding of the virus to other negatively charged molecules. Enhancing the stability of this complex would be expected to lead to reduction of the average minimal infective dose of inhaled virus particles, i.e. the minimal number of virus particles that is required to start an active infection. The same must also be expected to happen as a consequence of reduced non-specific binding of the virus to other negatively charged molecules, while the stability of the virus–receptor molecule remains constant.

Most likely, it may be enough with one virus particle productively infecting one cell in order to start a chain reaction leading to active infection. But since many virus particles probably will bind to negatively charged macromolecules other than the hemagglutinin receptors sitting on cell surfaces, and it is known that negatively charged macromolecules can interfere with influenza virus uptake by the cells [15], [16], it may be possible that on average a number of virus particles much larger than one is required in order that one virus particle be taken up by one cell and start to multiply there. At the same time, it must be expected that more efficient virus uptake by the cells also must lead to more rapid progression of the disease and a tendency for enhancement of the viral load, which may in turn lead to a tendency for aggravation of disease symptoms and enhancement of the probability that the patient will die [1], [7]. The observation from the Canton of Geneva that there was probably a very substantial enhancement of the infectivity of the virus from wave 1 to wave 2 [9], [10] is then highly compatible with the well-documented observation that there was also a very substantial enhancement of the lethality of the virus, comparing wave two with wave one.

It may be considered more likely than not that the same will also happen with hypervirulent H5N1 influenza, in the case that the virus should manage – without reassortment with other and less virulent influenza virus strains – to enhance its human-to-human transmissibility enough to start a pandemic. This gives us a gloomy scenario indeed, if the starting point (before the occurrence of mutations leading to a combination of enhanced human-to-human transmissibility with enhanced virulence) is a virus already possessing a lethality of 82% among confirmed human cases. While the Black Death in Europe left a population of survivors amounting, after all, to about 40% of the original population, it might be speculated that after an H5N1 pandemic starting in Indonesia, there might perhaps be only be some 4% of the original population surviving, considering those countries who are at a similar level of economic development to Indonesia before the start of the pandemic. And for those poor countries where the average diet is even poorer, it may be possible that the proportion of survivors would be even less (say, 4 per mille of the original population rather than 4%). In the rich countries (in the absence of vigorous counter-measures), it might be speculated that the proportion of survivors could be anywhere in the range from 90% (meaning that nearly half a million people would die even in a small country like Norway) to only 10%. The uncertainty regarding the possible pathogenicity properties of a hypervirulent H5N1 pandemic virus is so large that more accurate prediction is not possible; however, we should not forget that the worst case scenario would be something having 100% lethality among those infected all over the world (in the absence of previous immunization or treatment methods much better than what is available today). The higher boundary for the estimate of the proportion of survivors given above is found by multiplying average mortality during the Spanish flu pandemic by a factor of 20.

None of these figures should, of course, be taken as representing accurate and reliable predictions; they are only meant to illustrate, by order of magnitude, the scale of the possible disaster that we should now do our utmost to avert. It means, however, that we should not exclude the possibility that we might be facing, even in the rich part of the world, something that might turn out to be comparable to the Black Death in terms of mortality (i.e. deaths as proportion of the total population) – unless highly vigorous counter-measures are taken to avert it before it is too late.

However, even if the WHO no longer considers the Spanish flu pandemic to represent a valid worst case scenario, there are few indications that the new message has reached a majority of those politicians who are sitting in the governments and parliaments all over the world. It is, for instance, not possible to see from the Norwegian state budget for 2007 that the Norwegian Parliament could have been informed about the new signals from the WHO during the period they were handling the government's budget proposal (from October to December 2006). It is also extremely unlikely that members of the Norwegian government deliberately kept this kind of information back from the Parliament. The most likely explanation is that there has been a break in the chain of communication somewhere at a bureaucratic level; i.e. those in the bureaucracy may have failed to recognize the importance of the new message now coming from the WHO, and therefore have not found it necessary to tell the Norwegian government about it.

The world's practical preparedness for meeting something that might turn out to be comparable to the Black Death, if not even worse, does not appear to be much better now than when our article [1] was written last summer. It is still almost next to nothing. There has been one change for the better, since various governments are now trying to improve emergency preparedness by ordering and storing pre-pandemic vaccines. But nobody can know for certain how well these vaccines will work, if and when the pandemic comes. And the global production capacity for influenza vaccines is still only sufficient for a very small proportion of the total world population – at the same time as those populations that must be expected to stand as the last ones in the vaccine queue are the same ones for whom the mortality in the absence of vaccination would be expected to be highest.

So the title of our article [1] is, unfortunately, not at all outdated, even if the WHO has now come over to our side, so to speak, as regards their risk assessment. Nor is there any reason to consider our discussion of possible causes of poor emergency preparedness as outdated, neither when we discuss the possible role of communication failure through the signal chain from scientists via bureaucrats to politicians nor when we emphasize the probable role of psychological factors (‘the virus of malignant wishful thinking’).

Human vaccine development and poultry vaccination recommendations

A presentation from the WHO collaborating center in the UK reviewed the status of vaccine development at the WHO meeting in September [2]. It concluded that results to date have not been promising [2]. Efforts to develop a vaccine that confers adequate protection have been greatly complicated by the emergence of genetically and antigenically diverse viruses that are now simultaneously circulating in different geographical areas [2]. Vaccines that protected against viruses from clade one demonstrated poor cross-reactivity for virus subgroups in the second clade [2].

The results of several vaccine trials were reviewed at the meeting [2]. A number of different human vaccines are now available, and several clinical trials of candidate vaccines are under way in Europe, the United States, and Japan [2]. It is an obvious problem, however, that one cannot easily carry out clinical trials to test the efficacy of the vaccine against the real enemy, except through large-scale vaccination programs targeting tens of millions, if not hundreds of millions of people. The virus concerned is so highly pathogenic that experimental infection of volunteers is not feasible for ethical reasons. And the incidence of the disease is so low that one needs a very large test group and a very large control group to detect (as statistifically significant) any effect of the vaccine in relation to spontaneously occurring cases of H5N1 infection in humans.

Proxy indicators of effect, such as titers of anti-H5N1 IgGs, are of questionable value, given the limited protective effect of the vaccine types that have been traditionally used for protection against ordinary influenza [17]. For a virus that not only uses mucous membranes as the main route of entrance to the organism, but also has organs full of mucous membranes as the chief targets of infection, it appears obvious that it is not IgG immunity that is needed for efficient protection (except possibly for reducing the risk of infection of other organs such as the brain), but first of all IgA (and most likely also T-cell-mediated cellular immunity). But the traditional vaccine does not give any significant IgA immunity (because it is not administered in the right place in the body), and it is questionable (because it has not been well enough studied) how much T-cell-mediated protection it may offer. However, if the traditional vaccine gives only moderate protective effect against relatively non-virulent influenza virus strains, we should not be confident that this vaccine type will be good enough for efficient protection against an enemy that is orders of magnitude more aggressive and dangerous. Even a significant, but still moderate level of protection, e.g. reducing mortality by 50%, might in this case be far from the level of protection that we would ideally desire (and most laymen probably would expect) from a good vaccine. Of course, 50% reduction of mortality is much better than nothing, but still far from good enough when what we actually desire is something giving more than 90% protection.

The WHO report [2] raises several questions in connection with the use of pre-pandemic vaccines, which seem to reflect not only the problem of antigenic diversity of different clades and subclades of H5N1 avian influenza now circulating, but also a more fundamental uncertainty stemming from the lack of testing of the different candidate vaccines in a realistic combat situation against the real enemy. To quote the words of the report:

‘Participants noted that many fundamental questions underlying the development of an effective pandemic vaccine have not yet been answered. What is the best adjuvant for boosting the response? Does one antigen dominate in inducing antigen recognition? What are the benchmarks for assessing an adequate level of protection? In the absence of scientific answers to these questions, concern was expressed that national policy decisions about which vaccines to stockpile may be premature, despite the understandable desire of governments to invest now in some means of protecting their populations in the event of an influenza pandemic.’

The situation may be completely different in the field of vaccines used for animals for the simple reason that such vaccines can much more easily be realistically tested. The efficacy of poultry vaccination is demonstrated by the case of Viet Nam, where human cases of H5N1 influenza disappeared after the country had introduced a policy of mass poultry vaccination [2]. The WHO report also recommends a policy of poultry vaccination, attended by systematic monitoring, in such cases when culling is impracticable as a control strategy, in the interest of reducing opportunities for human exposures and infections to occur [2].

The WHO report does not, however, address the problem of enhanced selective pressure for antigenic drift of viruses circulating in animal populations as a direct consequence of vaccinating the animals [7]. Even if vaccination must be expected to reduce the prevalence of highly productive infection in the animals, which will in turn reduce the risk of virus transmission from animals to humans, it must also be expected to have the highly undesirable side effect of enhancing the rate of evolutionary change of antigenic properties for the virus – which may not only cause this evolution to proceed much faster than otherwise might have been expected (other factors being equal), but also might have the potential of enhancing the rate of evolutionary diversification of the virus (into a greater number of subclades). This, in turn, will make it more difficult to make enough different pre-pandemic vaccines, and it will also enhance the risk that none among the prefabricated vaccines will fit the actual pandemic virus when the pandemic starts.

These are good arguments for choosing good monitoring and culling rather than vaccination as the preferred method of controlling the disease in domestic animal populations. But there might be countries where the infrastructure is so poor or the situation so difficult for political reasons (civil war) that monitoring at the level of efficacy required here may be considered almost impossible. Such situations create a cruel dilemma, where it is difficult to oppose WHO's current recommendation in favor of poultry vaccination [2]. The only good long-term solution to this dilemma would probably be the development not only of vastly enhanced production capacity but also vastly improved flexibility of the global system of influenza vaccine production, so that an appropriate, cheap, and efficacious vaccine can be mass produced almost immediately as soon as a new dangerous virus strain needing a new vaccine has been gene sequenced.

Antiviral drugs – better in combination with other treatment methods?

There is a section in the WHO report dealing with antiviral drugs, but nothing is said about other possible methods of treatment, such as immunonutrition, or those other possible methods of treatment that were mentioned in our survey article about the role of nutritional factors in connection with hypervirulent avian influenza [6]. It is mentioned in the WHO report that a fair proportion of isolated H5N1 viruses are amantadine-resistant, ranging from 10% to 100% dependent on year and geographical location, with the exception of Qinghai Lake-like viruses that mostly show susceptibility to amantadine [2]. Resistance to the neuraminidase inhibitor oseltamivir (also known as Tamiflu) is less common than for amantadine, but resistance has been observed in a few patients, and that finding is considered to be of concern [2].

However, it was reported in a review article in the Lancet in January 2006 that no significant protective effect against lethality has been observed when oseltamivir has been used for treatment of human cases of H5N1 influenza in Southeast Asia [18]. It may be possible that this could be interpreted as a consequence of the mechanism of action of the drug, as seen in relation to the kinetic and regulatory aspects of viral replication in patients suffering from H5N1 infection in its present form. The most important problem with this virus in humans is apparently not its rapid replication (which might on the contrary be slower than for ordinary influenza virus, judging from its alleged longer period of incubation), but rather that a negative feedback regulatory mechanism is lacking (or grossly impaired), with the consequence that the viral load continues to grow and grow long after a normal influenza virus would have reached some plateau level controlled by the negative feedback regulatory mechanism(s) [1]. Giving a drug which reduces the rate of viral replication even more might then have the consequence that it will take a longer time for the viral load to grow to an excessive and potentially lethal level, but it will not hinder the virus population in continuing to grow long after a normal influenza virus population would have stopped (i.e. would have reached some plateau level). It may take some few more days before the patient dies, but the final outcome could still be the same.

Slowing the rate of progression of the disease may not, however, be without value provided that the near-terminal phase can be sufficiently delayed that the organism may manage to develop an adaptive immune response strong enough to fight back the enemy before death occurs. The likelihood that this may happen must theoretically be expected to depend very strongly on the nutritional status of the patient. If proliferation of leukocytes (or their progenitors) is too much inhibited because of specific nutrient deficiency conditions, there may presumably not be much hope that a patient infected with some hypervirulent avian virus will be able to survive. However, with optimal nutrition (with enough of everything that is needed for the leukocytes to grow), it may be expected that chances of survival will be better – and it may be possible that drugs such as oseltamivir may help to give the immune system the extra time it may need to win the race. So even if oseltamivir does not have any strong effect on the mortality of H5N1 patients when used alone (as monotherapy), it may be possible that it could have a much larger effect as part of an integrated, multifactorial therapeutic strategy where everything possible is done to try to enhance the rate of development of specific immune responses (dependent on B lymphocytes and T lymphocytes) at the same time as one also tries to slow down replication of the virus as much as possible. Immunonutrition (i.e. optimal nutrition for the immune system) should be seen as fundamental for this kind of integrated (and multifactorial) therapeutic strategy, but it could be supplemented not only with antiviral drugs, but – perhaps even more importantly – also with various immunostimulating agents, e.g. beta-glucans or immunostimulating hormones (see the section about new therapeutic options for more detailed explanation and references).

It may also be theoretically expected that drugs slowing the replication of influenza virus can be of greater therapeutic value than now against H5N1 viruses in a situation following evolutionary changes in the virus that help to improve the kinetics of its cellular uptake and thus may cause the disease to progress much more rapidly than has been common so far. The possibility should therefore not be excluded that oseltamivir might prove to be of considerably greater value (at least if used as part of a multifactorial therapeutic strategy) in a pandemic situation, when the virus may be expected to multiply more rapidly, than has been the case with H5N1 patients in Southeast Asia so far.

It might be speculated that larger average use (per patient per day) or earlier use of oseltamivir, comparing the situation in 2005 with that in 2003, possibly might help to explain the reduction of mortality among confirmed cases worldwide that took place from 2003 to 2005. However, this form of drug therapy seems not to have been efficacious enough to prevent the marked resurgence of high mortality that took place in 2006 compared with 2005.

New epidemiological observations from the Spanish flu pandemic

A very important paper about the Spanish flu pandemic appeared in the Lancet in the last issue in 2006 [5]. All countries with high quality vital registration data for the 1918–20 pandemic were identified, i.e. those countries in which registration is believed to be 80% or more complete. Twenty-six different countries were included in the investigation, while the USA also was divided into separate states (including only those 24 states where high quality registration data are available) and India into nine separate regions. Pandemic mortality rates for the UK, France, and Finland for 1918–20 were based on females only, since male mortality is confounded by deaths due to war. These data were next used to calculate excess mortality. Least squares regression models were developed that related excess mortality to per-head income and absolute latitude. Absolute latitude was tested because of arguments in the 1920s that environmental factors such as temperature were a key determinant of mortality [5].

No correlation of mortality with geographic latitude was found, but a strong negative correlation was found between excess mortality and per-head income [5]. A 10% increase in per-head income was associated with a 9–10% decrease in mortality [5]. Differences in per-head income accounted for about half of the total geographic variation in excess mortality [5]. The excess mortality data show that, even in 1918–20, population mortality varied over 30-fold across countries, comparing the region in India with highest mortality and the state in USA with lowest mortality [5]. A simulation was also carried out to see what would have happened today if there had been a new pandemic with a virus having properties identical with those of the Spanish flu virus and with the same age distribution of mortality as during the Spanish flu pandemic. It was found that an estimated 62 million people would have been killed (10th–90th percentile range 51 million to 81 million) and that 96% of all deaths (95% CI: 95–98%) in this hypothetical scenario would have occurred in the developing world [5].

The conclusions of this paper, concerning the great difference in excess mortality comparing more affluent and poor countries, agree with the data presented earlier by Johnson and Mueller [4]. Their paper is a compilation of the results of several historians working with available historical source data from several different countries; they include several countries not mentioned in the more recent Lancet article because the countries concerned do not have high quality vital registration data for the period 1918–20. Some of the countries mentioned in Johnson and Mueller's article had an estimated mortality outside the range of variation found in the study carried out by Murray et al. [5]. Even if the quality of these data may be questioned, it is not unreasonable that some of the countries in sub-Saharan Africa may have had an excess mortality even higher than India (up to half of the population was estimated for one of the countries in sub-Saharan Africa) and that the true range of geographic variation of mortality could be even higher (more than 100-fold?) than the factor of more than 30-fold found by Murray et al. [5].

There seems to be no plausible explanation for such large geographic variation of excess mortality during the Spanish flu, and for the negative correlation between excess mortality and per-head income, except differences in nutritional status comparing the populations in different parts of the world [7]. (See Thomas McKeon, The Modern Rise of Population [19] for a similar argument to explain the rapid decline of childhood infectious disease mortality in Europe during the period 1860–1970.) Several possible reasons for this have been explained in an earlier survey article [6]. The mechanism and kinetics of dispersal of the influenza virus mean that geographic differences in mortality during the Spanish flu pandemic cannot be explained as a consequence of geographic differences in hygienic factors [7], except to the extent that differences in hygienic factors may have contributed to geographic differences in the nutrition status of the population (because of the impact of hygiene-related parasite infestations and infectious diseases on nutritional status [20], e.g. the role of hookworm and schistosomiasis as causes, alone or with other factors, of iron deficiency). Since high mortality affected several ethnically and genetically highly diverse groups [5], it appears unlikely that genetic differences between different populations could have played any major role [7]. Differences in the age structure of the population may, however, have played a role, given the curious W shape of the curve describing the average mortality rate of different age groups as a function of age [5], [8]. But this factor can only account for a minor part of the total geographic variation in excess mortality.

It is well known, however, that not only the total per capita food intake, but also the average composition (and hence nutritional quality) of the diet is strongly dependent on per-head income. One major reason for this is the higher average cost of production (per unit of food energy) of more protein-rich plant foods compared with plant foods poor in protein (e.g. cassava roots, cane sugar), and the negative correlation found between protein/food energy ratio and food energy production per hectare per year, comparing several different plant crops important as sources of food energy and/or plant protein. Legumes contain more protein, but have mostly lower food energy yields (per hectare per year) compared with various cereal crops, and the latter in turn contain more protein, but have lower food energy yields (per hectare per year) compared with cassava roots, cane sugar or palm oil. And animal protein foods are in most cases even more expensive to produce (and give even less food energy per hectare per year) compared with protein-rich plant crops such as soybean and other legumes.

The availability of enough good quality dietary protein may be very important, however, for the chance of surviving infectious episodes caused by dangerous pathogens such as the more virulent strains of influenza virus. One of the main reasons for this is the special role of the non-essential amino acid glutamine as a favored energy nutrient for many leukocytes [6] at the same time as normal synthesis of proteins in the cells (which is needed for normal cellular growth) depends on an adequate supply of all essential amino acids including lysine, which is the most common limiting amino acid in monotonous cereal-based diets, and tryptophan which is often also limiting in monotonous diets based on traditional varieties of maize – at the same time as interferon (IFN)-γ can induce induction of the tryptophan-degrading enzyme indole-2,3-dioxygenase [6]. Another reason is the importance of glutathione (and hence of the dietary intake of sulfur amino acids) as a cofactor used during DNA synthesis, as an anti-apoptotic agent, and for controlling the balance between Th1-associated and Th2-associated cytokine secretion [6]. Other nutrients important for immunological functions, such as zinc [21] and nucleotides in the form of RNA and DNA (because of their role as precursors for endogenous nucleotides through various salvage pathways), are also strongly associated with protein in the diet. Iron is found both in plant foods and animal foods [21], but intestinal absorption is mostly much better for iron coming from animal foods than for iron coming from cereals or legumes. Vitamin B₁₂, which is important for DNA synthesis, for the function of cytotoxic CD8⁺ T cells and for the control of cytokine (tumor necrosis factor (TNF)-α) secretion [6], is found nearly exclusively in animal foods (with the exception of marine algae and some fermented food preparations). The same is also the case with the aminosulfonic acid taurine, which will be more closely discussed in the section dealing with possible new therapeutic modalities for treatment of hypervirulent influenza.

Note also, that traditional and modern products made from whole fish, such as kapenta and fish protein concentrate type B, may be considered exceptionally well-balanced sources of multiple essential and conditionally essential nutrients that are more typically associated with animal foods (because of larger abundance or better intestinal absorption) than with plant foods. If made from cheap marine raw material, such as pelagic or mesopelagic fish associated with areas of oceanic upwelling, they are most commonly also good to excellent sources of several nutrients more typically associated with seafoods (e.g. iodine, selenium, vanadium, long-chain omega-3 fatty acids, and taurine) at the same time as they can be produced more cheaply, comparing production costs per kg protein, than practically any other animal foods.

Geographically variable nutritional factors may explain much of the geographic variation in mortality during the Spanish flu pandemic

But there are also very important geographical variations in diet composition that are not directly related to per capita income. This is of course the case with several soil factors, e.g. concentrations and bioavailability (for uptake into the plant roots) of elements such as iodine, selenium, sulphur, and zinc in the soil, which may in turn depend on several different factors such as bedrock composition [22], [23], intensity of chemical weathering processes [24–26], annual rainfall, topographic relief, distance from the sea [22], [27–30], frequency of anthropogenic fires [31], [32], etc. There are also large geographic variations, not directly related to per-head income, concerning the use of several different staple foods with notably different composition (e.g. whether people eat mostly rice, maize, wheat, millets or cassava roots). Moreover, there are also important variations in cooking habits, which affect the composition of the diet (e.g. its concentration of heat-labile micronutrients such as folate, or the concentration ratio of phytate to zinc) and which may depend strongly on the local cultural traditions (e.g. whether people prefer fermented or non-fermented bread types, or whether they prefer fermented or non-fermented legume preparations), but not on per-head income as such. Observational studies have shown some of the more typical soil-dependent factors such as selenium to be extremely important for survival among patients with HIV infection [6], and there may be good reason for suspicion that something similar might be the case for hypervirulent influenza as well [6]. It may therefore be possible that geographical differences in diet composition also might help to explain much of that part of the geographic variation in excess mortality during the Spanish flu pandemic which cannot be statistically accounted for as related to per-head income.

The mineral nutrient composition of plant tissues appears, generally speaking, to be less well physiologically regulated than that of animal tissues, even though there are certain mineral nutrients (among the essential trace elements) that are also very poorly regulated in animals. The mineral nutrient composition of plant foods, e.g. cereal grains, is therefore more sensitive to geographic variations in the chemical and mineralogical composition of the soils which, as already mentioned, depends on several different factors. This means that there is much hidden variation in the composition of plant foods, which is not reflected in the commonly used food composition tables. It means that it is not enough to measure the amounts of different types of food eaten and use a food composition table for calculating the intake of different mineral nutrients unless one has a method of making some kind of local calibration of the data found in the food composition table.

If nutrition variables can account for geographical variation of excess mortality during the Spanish flu pandemic by perhaps a factor of 10 or more, even when comparing the average figures for large populations, it must be expected that the range of variation comparing individuals must be even larger (considering the death risk for each individual as a function of nutrition status). This contrasts greatly with the very limited effect of oseltamivir on mortality when used on patients with H5N1 influenza in Southeast Asia [18]. Why then is the role of nutrition factors not considered at all (or at least not mentioned in the report after the meeting) when WHO convenes a meeting including several of the foremost influenza experts in the world, as it did in September last year? And why is it not emphasized (possibly in most cases not even mentioned) when different WHO member countries make their national pandemic preparedness plans? It is not easy to find any other plausible explanation, except the problem of over-compartmentalization being a scourge not only for far too much of modern medical science, but also for more than we like to think of when considering our practical medicine whether at a therapeutic or a prophylactic level. The British neurological scientist F.M.R. Walshe made a comparison with the evolution of the central nervous system in vertebrates in an important essay addressing this problem, when he quoted Sherrington's dictum: ‘Integration keeps pace with differentiation’ [33]. While this is an ideal model for how things ought to be in the realm of medical research, reality is unfortunately – says Walshe – completely different. There is no strong reason to believe that the situation has become much better today than it was in 1948, when Walshe's book was published.

The present authors have previously given several theoretical reasons why nutrition factors may be very important for the chance that an individual may survive an attack by some highly pathogenic strain of influenza virus [6]. These theoretically based predictions seem to be in excellent agreement with the high quality observational data now available [5].

Age distribution of mortality during the Spanish flu pandemic: possible explanations

The Spanish flu article by Murray et al. [5] also has a figure showing the variation of age group-specific mortality for men and women as a function of age. The curve only has a time resolution of 5 years, but it shows a steep decline from 0–4 years to 5–9 years, a modest decline from 5–9 years to a minimum at 10–14 years, from where the curve rises steeply to reach an acute peak at an age somewhere between 25 and 30 years [5]. The curve then falls first fairly steeply and then more gradually until another minimum (but now a much flatter one) is reached somewhere in the interval between 50 and 60 years [5]. The curve also shows that the average mortality was less for women of fertile age than for men of the same age, while the mortality for old men and old women was the same [5]. The sharp peak on the curve is seen at the same age (within the time resolution of the curve) both for women and men [5].

What does this mean? It is reasonable to interpret the shape of the curve from 0 to about 28 years as being controlled mainly by age-related endogenous factors, such as age-related changes in the levels of immunostimulating hormones [7], and maybe also mitochondrial DNA aging. But it is not plausible to explain the sharp peak on the curve and subsequent steep decline (for ages >30 years) as a consequence of endogenous factors alone. There is no hormone behaving that way, with a sharp minimum or maximum (when considering the level of the hormone as a function of age) at an age between 25 and 30 years both for men and women. Nor is it easy to imagine any other endogenous factor changing as a function of age (such as the number of mitochondrial DNA mutations) that shows similar behavior.

The only plausible explanation for the shape of the curve among young to middle-aged adult men and women (between 15 and 55 years old) is therefore to assume that the decline of the curve as a function of age for those more than 30 years old may be due to adaptive immunity – as has been suggested earlier [34] – directed against antigenically similar viruses that were circulating before the ‘Russian’ pandemic around 1890 [7].

This raises an important question about the (now obsolete) use of death figures from the Spanish flu pandemic as a worst case scenario. What would the mortality figures have been if not a majority of those more than 30 years old had been exposed to antigenically similar viruses before, and therefore benefited from excellent anti-Spanish flu adaptive immunity? And what would have happened if the more virulent (and more infective) wave two virus had come first, instead of the actual wave one virus? In both cases, there is strong reason to suspect that mortality easily could have turned out to be much higher than was actually seen. The Spanish flu virus may have been even worse than its reputation.

Hypervirulent influenza pathogenesis and possible implications for understanding the attack strategy of the virus

The report from the WHO September meeting [2] summarizes what is known about the typical clinical course of human H5N1 disease as follows:

‘The disease caused in humans by the H5N1 virus was described as fundamentally different from that caused by normal influenza. In H5N1 infection, the disease syndrome typically shows progressive primary viral pneumonia, acute respiratory distress, marked leukopenia and lymphopenia, and (in some cases) diarrhoea and liver or renal dysfunction. What might explain this severity? Some limited findings suggest that the virus might cause disseminated infection, affecting multiple organs. In some patients with a fatal outcome, virus has been detected in faeces, serum, and blood plasma. However, respiratory pathology remains the primary cause of death. Additional data presented support the hypothesis that severe disease is based on the induction of a “cytokine storm”; it was pointed out, however, that this remains a “chicken-and-egg” dilemma – does an overwhelming level of cytokinemia result in, or from, excessive tissue damage and disease?’

A very important recent report (from January 2007) deals with experimental H1N1 influenza infection in cynomolgus macaques (Macaca fascicularis), comparing an ordinary low-virulent H1N1 strain with the Spanish flu virus [35]. The results of this study are judged by the present authors to be so important, especially when compared with what is known about H5N1 infection in humans, as quoted above (but also when compared with what is known about H5N1 infection in experimental animals), that a detailed account of the most important observations is given below. An attempt will be made to analyze this information in order to understand better the attack strategy used by the enemy (to explain why mortality during the Spanish flu pandemic was so high, and why it is so high among human cases of hypervirulent H5N1 influenza). This ‘military intelligence’ information regarding the main attack strategy used by the enemy will subsequently be used for making a defense strategy that hopefully can work, provided that the virus is not too super-aggressive and provided that efficacious weapons that can be used as part of this defense strategy have both been adequately tested and produced in such quantities that may be needed in a real war situation, i.e. if or when a pandemic with hypervirulent avian influenza has already begun and is spreading around the world without being stopped effectively enough.

The 1918 virus caused a highly pathogenic respiratory infection that culminated in acute respiratory distress and a fatal outcome [35]. Furthermore, infected animals mounted an immune response that was characterized by dysregulation of the antiviral response [35]. The antiviral immune response was insufficient for protection, but there was also a high production of various cytokines, such as interleukin (IL)-6 and IL-8, which in an important way may have contributed to host tissue pathology [35]. These data are thought to indicate that atypical host innate immune responses may have contributed in an important way to lethality [35].

All Spanish flu-infected animals became symptomatic within 24 h post-infection [35]. They appeared depressed, were hesitant to eat normal food and to drink, and showed respiratory complications with nasal discharge and non-productive cough [35]. They became progressively more debilitated and eventually developed an acute respiratory distress syndrome [35]. Respiratory signs were the most pronounced indication of illness, with increase of respiratory rate by an average factor of about 3 on day 8 of infection [35]. Decreases in lung function shown by a decrease in blood oxygen saturation of as much as 36% compared with pre-infection levels were detected by pulse oxymetry [35]. Consistent changes in heart rate and blood pressure were not observed [35]. In contrast, animals infected with the ordinary influenza virus showed only few and very mild clinical signs [35].

The 1918 virus was present at high titers in both the upper and lower respiratory tissues on days 3, 6, and 8, while the ordinary influenza virus was isolated mainly from the upper respiratory tissues on days 3 and 6 and on day 8 in only one tonsil [35]. The 1918 virus was also recovered from the heart and spleen of some animals, but neither virus was isolated from the brain, kidneys, liver or colon of any animals [35]. Lungs of the 1918 virus-infected animals were the only tissues to exhibit macroscopically observable pathological changes as severe lesions, with 60–90% of the lung tissue affected by days 6–8 [35]. Profuse watery and bloody liquid filled infected areas, greatly reducing lung function [35].

By days 6 and 8, the lungs of ordinary influenza-infected animals showed signs of healing, evidenced by thickening of the alveolar wall and no viral antigen expression [35]. In contrast, the lungs of all 1918 virus-infected animals showed worsening alveolar damage and substantial viral antigen [35]. Extensive edema and hemorrhagic exudates were prominent, as reported for human patients who succumbed to the ‘Spanish’ influenza in 1918–20 [35]. By day 8, expression of viral antigen was diminished or undetectable in consolidated alveolar areas, but still appreciable around consolidated areas [35]. Bronchiolitis and bronchitis with expression of viral antigen were prominent at this time [35].

It may be added that these observations of pulmonary pathological changes in Spanish flu-infected macaques are very similar to what has been observed in mice experimentally infected with highly pathogenic avian H5N1 virus – where highly edematous lungs with inflammatory cell infiltration and alveolar and interstitial edema also were found [36], along with hemorrhage in the lungs, progressive and severe hypoxemia, and significant increase in neutrophils, TNF-α, and IL-6 in bronchial alveolar lavage fluid [36].

Among the cytokines and chemokines tested in the Spanish flu virus-infected macaques, substantial increases of IL-6, IL-8, CCL2 (monocyte chemoattractant protein-1), and CCL5 (RANTES) were detected in the sera of infected animals [35]. No changes in IL-2, IL-4, IFN-γ, or TNF-α were found [35]. Both IL-8 and CCL5 were elevated to similar levels in animals infected with either virus [35]. The most striking changes were seen with IL-6 [35]. In 1918 virus-infected animals, IL-6 increased 3–9-fold by day 3 post-infection, 6–19-fold by day 6, and 5–25-fold by day 8, while no significant change in the IL-6 level was detected in animals infected with ordinary influenza virus [35]. In humans experimentally infected with influenza virus, it has been reported previously that IL-6 expression at both the site of replication and in sera directly correlated with the extent of viral shedding, and it is thought to have a role in mediating the clinical manifestations of infection [37], [38]. However, TNF-α is also expressed during experimental human influenza infection, even though peak expression of this cytokine is delayed compared with the peak of the viral titer and the peak of IL-6 expression [37].

Global gene expression profiles in the bronchi of infected animals were also studied, using microarray analysis [35]. One striking observation was the relative constancy in gene expression profiles in the 1918 virus-infected animals at days 3–8 post-infection, in marked contrast to the ordinary influenza-infected animals, which showed an appreciably more dynamic response during the course of infection [35]. Animals infected with ordinary influenza virus showed a marked increase in expression of mRNAs for various type I IFNs and a corresponding increase in mRNA expression of genes stimulated by type I IFNs early in infection (and coinciding with the greatest load of the virus) [35]. This response was down-regulated on days 6 and 8 post-infection, when the virus was no longer detected [35].

The 1918 virus-infection, by contrast, induced much fewer IFN-α genes, suggesting that it caused an altered antiviral response in the bronchus [35]. The 1918 virus-infected animals also showed differential activation of type I IFN-stimulated gene expression, despite viral titers in the bronchi that were 10–5000-fold higher than in the animals infected with ordinary influenza virus [35]. The gene expression profiling data suggest that the 1918 virus induces an antiviral response different from that of ordinary influenza virus, possibly including reduced sensitivity to type I IFN responses [35].

It is mentioned in the same article that an important pathway in the activation of the antiviral response to influenza virus infection occurs through the activities of DDX58 (or retinoic acid-inducible protein I) and IFIH1 (or melanoma differentiation-associated gene) [35], [39], [40]. DDX58 is also known under the name RIG-I [39]; it is a cytoplasmic helicase protein that detects in vitro transcribed double-stranded RNAs (dsRNAs) [39]. IFIH1 is also known by the name MDA5 [39]; it is another cytoplasmic helicase protein that also detects dsRNAs [39]. But these two proteins recognize different types of dsRNAs; IFIH1 recognizes poly(I:C), and RIG-I detects in vitro transcribed dsRNAs [39]. RNA viruses are also differentially recognized by RIG-I and IFIH1 [39]. RIG-I is essential for the production of IFNs in response to RNA viruses including paramyxoviruses, influenza virus, and Japanese encephalitis virus, whereas IFIH1 is critical for picornavirus detection [39]. Since retinoic acid plays a role in the regulation of the expression of this protein (as implied by the name retinoic acid-inducible protein I), it might be speculated that impaired expression of RIG-I could be one of the main reasons for the more virulent behavior of certain viruses that can be observed in patients suffering from vitamin A deficiency. Theoretically it would be expected that this might be the case also with ordinary influenza. RIG-I gene expression is activated by TNF-α [40], and it was later reported that the 5′-triphosphate end of RNA generated by viral polymerases is responsible for RIG-I-mediated detection of RNA molecules [41]. Detection of 5′-triphosphate RNA is abrogated in eukaryotes by capping of the 5′-triphosphate end or by nucleoside modification of RNA, both occurring during post-transcriptional RNA processing [41]. Uncapped 5′-triphosphate RNA (now termed 3pRNA) is present in viruses known to be recognized by RIG-I, but absent in viruses such as the picornaviruses that are known to be detected by IFIH1 (MDA-5) [41]. RIG-I-deficient and IFIH1-deficient (double knockout) mice are highly susceptible to infection with these respective RNA viruses compared with control mice [39].

The genes for RIG-I and IFIH1 were found to be induced in the ordinary influenza-infected, but not in the 1918 virus-infected animals [35]. This may help to explain why the type I IFN response is so strongly impaired in macaques infected with Spanish flu influenza virus [39]. At the same time, it may also be theoretically expected that infection with Spanish flu virus (and H5N1 virus?) will make macaques and presumably also humans extremely susceptible to simultaneous infection with other viruses that normally would be recognized by either RIG-I or IFIH1, such as picornavirus, Japanese encephalitis virus, and paramyxoviruses. However, it has recently been reported in another article that influenza A virus infection does not generate dsRNA and that RIG-I is instead activated (during infection with normal influenza virus) by viral genomic single-stranded RNA (ssRNA) bearing 5′-phosphates [42]. The mechanism of RIG-I activation in normal influenza may therefore be slightly different from what is suggested by the authors of the macaque Spanish flu article [35].

It is further mentioned in the macaque/Spanish flu article [35] that the non-structural protein 1 (NS1) of virulent influenza viruses can modulate the IFN-mediated antiviral response [43–45], and RIG-I is a target of NS1 immunosuppressive activity [39], [42]. It was recently reported in another article [46] that the NS1 protein of influenza A virus inhibits RIG-I-mediated induction of IFN-β. This happens because NS1 inhibits the activation of transcription factors, including IRF-3, that are involved in IFN-β transcriptional activation [46]. The observations suggest that RIG-I, IPS-1 (which is a downstream signaling partner of RIG-I) and NS1 become part of the same complex and that when binding to RIG-I, NS1 inhibits the downstream activation of IRF-3, thus preventing the transcriptional induction of IFN-β [46]. There are, moreover, other recent research reports (from other groups of scientists) that confirm these observations [47], [48].

While these observations may help to explain the high lethality of the Spanish flu virus in humans, it may be possible that they also could throw new light on the evolution of virulence properties in influenza type A viruses in general. It may be possible that we should not regard non-virulence as the phylogenetically more primitive trait and virulence as a derived character, but rather the opposite: that a potent anti-immunological weapon, which will serve as a high virulence trait whenever it is fully activated, could be something that belongs to the normal genetic make-up of this group of viruses. What may have happened in the ‘normal’ and relatively non-virulent strains of influenza type A viruses is that this anti-immunological weapon for evolutionary reasons has become temporarily silenced. The reason why it does not disappear once it has been silenced might perhaps be that it has also acquired other functions that are important for the survival even of non-virulent strains of influenza type A, and that it continues to carry out these other functions even after having been silenced (or de-activated) as a potent anti-immunological weapon. The knife is still being used for cutting bread, even if it is not used as a weapon any more. Or perhaps there is only a down-regulation, but not a total inactivation of its function as an anti-immunological weapon. It might still be active enough to be evolutionarily preserved. But only modest evolutionary changes (i.e. a modest number of mutations) might be enough for this anti-immunological weapon to be fully reactivated. Which means that we might compare the virus with a psychopathic and very dangerous criminal who is endowed with enough social intelligence to understand that for the time being, it is more advantageous for him not to commit any form of serious crime – but who is ready at the slightest notice to revert to his criminal ways as a murderer and a rapist, once the external conditions change (e.g. because of civil war, or because he has become a dictator), so that he can do these things without any risk of punishment.

A hypothesis of this sort might help to explain why there have been so many different recorded cases of appearance of high virulence traits in influenza type A viruses in domestic animals and also the short time needed for evolution of a low virulence viral strain into a high virulence strain. In 1878, a disease which very likely was some kind of influenza type A was identified in animals in Italy by Eduardo Perroncito [14]. He described an initially mild disease in domestic birds that after a while turned highly pathogenic, killing virtually all birds in the area [14]. But this is not the earliest historically recorded case of disease most likely being the same as highly virulent influenza. An epidemic of what was most likely influenza was associated with high mortality both in Europe and North America during the mid-16th century AD [1], and it may be possible that it had already been encountered in the Bible in the history about a deadly disease that killed many Israelites in the Sinai desert (following their exodus from Egypt) after they had eaten dried (but uncooked?) meat from quails; this could possibly have happened more than 4000 years ago [1]. Between the years 1959 and 1999, 18 outbreaks of avian influenza with high mortality were reported in domestic poultry around the world [14]. These outbreaks had devastating economic consequences for the affected countries [14].

From the perspective of evolutionary biology, it might also be reasonable to speculate that this anti-immunological weapon (the NS1 protein) might not be exclusively associated with influenza type A viruses, but rather could be something that so to speak may have been floating around, so that it may been transferred horizontally between several different types of RNA virus since very ancient times, being perhaps almost as ancient as those molecules used for antiviral defense that it tries to neutralize.

But it may also be possible that there could be important differences comparing different host species, so that a given type of NS1 protein will not be able to function as a good anti-immunological weapon except in a certain subset of those species that can be infected by otherwise closely similar or even identical type A influenza viruses. Adaptation of the NS1 protein to function as a good anti-immunological weapon in this particular subset of species might possibly entail that the same function will be lost in another subset of influenza type A host species. There is no good evolutionary reason that the distinction between those species that are sensitive and those that are insensitive to a given type of NS1 protein should follow the natural phylogenetic boundaries (between different groups of animal species), except perhaps when dealing with closely related species belonging for instance to the same genus or the same subfamily. We might be dealing with another example of the principle that war between different types of organisms (whether between animals and several types of pathogens or between plants and the animals which like to eat them) probably should be regarded as one of the most important causes of evolutionary diversification, leading to enhancement of the total number of evolutionary lineages and species. Most likely it is also one of the most important evolutionary motors leading to evolutionary progression in the sense of complexity evolution (from less to more complexity). Heraklit may therefore have been right, in the sense of evolutionary biology and evolutionary history, when he claimed that the war is the mother of everything (and of course he was also right, in the sense of evolutionary biology, when he said ‘Panta rei’, which means: ‘Everything flows’). But this is a form of war more dependent on position (geographic location), situation, and opportunity than where in the great system of natural taxonomic classification the different combatants might belong.

It should be very much worthwhile to make a systematic comparison among several different avian and mammalian species of those intracellular proteins that are targets for the anti-immunological effects of the NS1 protein to see if one can find a pattern compatible with what is known about the virulence characteristics of the same type of virus in several different species of animals. Are the NS1-binding proteins more similar (as regards the stability constant of the complex between the intracellular host protein and the viral NS1 protein) when comparing humans and chicken (or humans and quail) than they are when comparing chicken and ducks, or humans with mice?

The new observations concerning the behavior of the Spanish flu virus in macaques imply that there is more similarity (more overlap) between the virulence properties of Spanish flu virus and hypervirulent H5N1 virus than we were aware of last summer 2006, when we wrote our article ‘Why is the world so poorly prepared for a pandemic of hypervirulent avian influenza?’ [1]. It is another example illustrating how review and commentary articles rapidly can become partly outdated when one is working in a field of very active international research, where important progress in understanding can take place almost from one month to another. The cause of our mistake was that we had assumed that observations that had been done with rodents might be valid in primates as well. But the experiment now carried out with macaques shows that this assumption was wrong.

The sum of all observations, especially concerning what happens during infection in humans, leaves very little doubt that hypervirulent H5N1 virus is far more dangerous for us, once we become infected, than was the Spanish flu virus. But it may be possible that the differences between hypervirulent H5N1 virus and the Spanish flu virus are mainly of quantitative rather than qualitative nature, so that one is dealing with differences of degree rather than of principle. We may possibly not be dealing with the difference between a one-headed and a three-headed monster [1], but rather with the difference between a three-headed monster child and a three-headed monster adult warrior.

The macaque experiment [35] seems also to indicate that the cytokine IL-6 may be far more important in hypervirulent influenza (as compared with other cytokines including TNF-α) than we were aware of when writing our earlier survey articles [1], [6]. It should be added, though, that there are other observations [37] showing that TNF-α can be much more important in human influenza than was observed in the macaque experiment. However, TNF-α does not appear to be indispensable for the highly virulent influenza to take a deadly course in primate species. IL-6 seems also to be capable of doing the same job, even though the main mechanisms of action of the two cytokines when acting as killer agents are most likely not the same, if one may conclude from those research data that are available today (which, however, may not necessarily represent the final word about this question).

Alveolar edema causing impaired oxygenation of arterial blood was observed to be very important in the pathogenesis of Spanish flu influenza in macaques [35]. It recalls the observations of cyanosis in human patients with an influenza-like illness that were already recorded from the war theater in France during the period 1915–1917, i.e. before the Spanish flu pandemic started in 1918 [49]. There may thus be good reason to ask the question, what possible role excessive IL-6 (or TNF-α) secretion might play in the causation of alveolar edema in macaques or humans.

It might be speculated that the most important mechanism here might not be related to a more direct action of the cytokines on cells in the walls of the blood vessels, causing enhanced leakiness of the endothelium and therefore enhanced export of plasma proteins into the extravascular tissue space. The development of edema following injury in other tissues, e.g. in the skin, seems to be controlled in large measure by nerve fibers (by secretion of various neuropeptides) rather than only by leukocytes (by secretion of histamine). It is not unreasonable to suggest that the same might also be the case in the lower airways. One may therefore ask the question: is it possible that we could be dealing with essentially the same phenomenon (but in another anatomical location) as when the skin swells locally following an insect bite or a burn, i.e. another case of neurogenic inflammation (cf. [50–53]) mediated by so-called axon reflexes in peripheral unmyelinated nerves?

It has been known for quite a long time that TNF-α can up-regulate the sensitivity of C-fibers (unmyelinated nerve fibers) important for the mediation of neurogenic inflammation [54–56]; recently it was reported that the same is also the case with IL-6 [57]. Injection of IL-6 and co-injection of IL-6 plus soluble IL-6 receptor (sIL-6R) into the knee joint of rats were found to cause a gradual increase in the responses of C-fibers to innocuous and noxious rotation within 1 h [57]. The increase in responses to IL-6 and IL-6 plus sIL-6R was prevented by co-administration of soluble glycoprotein 130 (sgp130), but sgp130 did not reverse established mechanical hyperexcitability [57]. Responses of Aδ fibers (which are thin myelinated nerve fibres) were not altered by these compounds [57]. While injection of sIL-6R alone into the normal knee joint did not influence responses to mechanical stimulation, injection of sIL-6R into the acutely inflamed knee joint was found to cause an increase in responses [57].

C-fibers can also be sensitized (or activated?) by reactive oxygen species [58–62], which will be produced by various types of leukocytes (such as monocytes, macrophages, neutrophils, and eosinophils) when they are activated because of infection or some other inflammatory process. Moreover, C-fibers can also be activated (or sensitized) by several different eicosanoids including PGE₂, PGF_2α, PGI₂, thromboxane A_2′ and leukotriene B₂ [63], [66–68]. Some of these eicosanoids are also produced in greater quantity during inflammatory processes than in corresponding normal tissues, especially as a consequence of up-regulation of the expression of COX-2 in leukocytes during inflammation.

It is therefore not at all unreasonable that a strong inflammatory reaction in the lower airways (as a consequence of excessive proliferation of influenza virus), which will be associated with the production of abnormally large quantities of a number of different C-fiber-activating or C-fiber-sensitizing substances, next might lead to abnormally strong activation of C-fibers in the same region, which could lead to hypersecretion of neuropeptides such as substance P, neurokinin A, and calcitonin gene-related peptide (CGRP) (cf. 51–53). Neuropeptides released from sensory C-fibers are in turn known from studies in various organs to cause vasodilatation, vascular congestion, and extravasation of liquid from the post-capillary venules, with resultant edema and exudate [69–75]. It has also been shown that some of them can cause plasma extravasation and edema in the lower airways [76–80]. There are, moreover, also observations from other organs suggesting that neurogenic inflammation may contribute to the development of local hemorrhage as a consequence of erythrocytes leaving venules throughout the endothelial junctions [81].

But IL-6 also has important direct effects on endothelial cells and can cause endothelial dysfunction [82]. It is therefore probable that the effects of IL-6 overproduction on blood vessels in the lower airways may be due to a combination of both the direct effects of this cytokine on the blood vessels and its indirect effects mediated by C-fiber sensitization or activation (in combination with other inflammatory mediators very likely including reactive oxygen species (ROS) and prostaglandins).

Possible role of nutritional factors in patients with H5N1 influenza or Spanish flu

When leukopenia and lymphopenia are observed in human cases of H5N1 infection [2], there can be little doubt that one must be dealing with a disease condition associated with severe immunosuppression. There must be good reason for hope that almost anything that might help to prevent or reverse the associated functional disturbances (i.e. helping to normalize immunological functions) might be of therapeutic value. But we need to address the question of what might be the main causes of the leukopenia and lymphopenia that have been observed in many human patients with H5N1 influenza? Does it happen because of scarcity of important nutrients needed for normal cellular growth? Does it happen because of enhanced leukocyte apoptosis that might also be partly related to some nutritional deficiency condition (but also the enhanced oxidative and nitrosative stress being a direct consequence of the immunological reaction to infection)? Or does it happen primarily as a consequence of dysregulation with an over-abundance of mainly immunosuppressive signal substances (such as PGE₂ and transforming growth factor (TGF)-β) or a relative deficiency of one or more critical signal substances needed for optimal stimulation of leukocyte growth?

It is not easy to obtain any reliable answers to these questions without very thorough clinical observations, where several different nutritional (or biochemical) and immunological parameters need to be studied simultaneously in the same patient. However, from all the experience that has been gained from clinical chemical and nutritional observations in connection with other infectious diseases both in humans and in experimental animals, it must be considered a highly plausible working hypothesis that the immunosuppression (with leukopenia and lymphopenia) that has been observed in human H5N1 cases may in large measure have a nutritional explanation – that we may at the same time be dealing with (i) cellular growth inhibition, (ii) a tendency for enhanced leukocyte apoptosis, and (iii) immunological dysregulation (because of overproduction of substances like PGE₂ or TGF-β), with all of these disturbances being in large measure a direct consequence of specific nutritional deficiency conditions such as for instance intracellular GSH depletion. But the deficiency conditions concerned may themselves have developed mainly as a consequence of the disease, not only because of the production of large quantities of cytokines (including TNF-α) with protein-catabolic effect (especially in skeletal muscle), but also because of excessive production of reactive oxygen and nitrogen species that will lead to enhanced oxidative degradation of various nutrient molecules that are relatively non-resistant against oxidative stress, such as folate [83], but also some of the other micronutrients, including selenium in the form of highly oxidation-labile [84] selenide ions. It may be noted that protein molecules are also vulnerable to oxidative damage and that this functions as one of the labeling methods that are used in the cell to target protein molecules for degradation by proteasomes, with oxidation targeting them for degradation by 20S proteasomes [85], [86], while ubiquitination is used for targeting them for degradation by 26S proteasomes [87].

If this working hypothesis is correct, it follows as a direct logical consequence what the therapeutic strategy must be. What should be considered most important is to try to correct all the specific nutritional deficiency conditions concerned, or feed the patient in such way right from the start of the infection that their development can be mitigated. This should probably be regarded as a necessary fundament for all other forms of therapy. But one may additionally also try to use other forms of immunostimulation, as will be discussed more later, e.g. in the form of immunostimulating hormones (which should be provided as cheaply as possible to be of any practical value for poor patients in poor countries), β-glucans or other substances having a similar mechanism of action (acting, for instance, via various Toll-like receptors) when they stimulate Th1 immunity and the innate immune response. It should be emphasized, though, that it may not be much use during a war to give orders to soldiers for attack when for several weeks they have had neither enough food to eat nor adequate ammunition for their weapons. So one should not hope for much effect from any kind of immunostimulant unless one has first given the leukocytes all the food they will need to solve their important and difficult task.

The experiments where macaques were infected with the Spanish flu virus [35] have demonstrated that a particular form of immunosuppression may play a pivotal role in the pathogenesis of hypervirulent influenza, at least in the case of Spanish flu virus infection, but most likely in the case of hypervirulent H5N1 infection as well, which is also in agreement with earlier observations on the latter disease [1]. This lesion hinders the immune system from targeting the enemy in the way it normally should have been done, but it does not hinder the development of other forms of immune reaction that primarily are harmful to the tissues of the host organism itself without being very effective in driving back the enemy. We should not think of this process primarily as representing some form of immunological over-reaction because the immune system is too strong. The problem seems rather to be exactly the opposite, i.e. a form of immunological dysregulation where the basic cause is a particular (but highly specific) form of severe immunodeficiency, and where many of the weapons still remaining are used in a particular form of suicide rather than for killing the enemy. This problem might be compared to a hypothetical situation in an ordinary war where a double agent has managed to infiltrate the military headquarters of the nation under attack, and where this agent manages to make a top general drunk before he starts to issue meaningless orders in the name of the same general with the consequence that the main artillery bombardment will now be directed against places with large concentrations of the general's own civilians instead of against the enemy forces.

However, even if a very important part of the antiviral defense system has already been taken out by the enemy, the situation might still not be hopeless since there are also other defense weapons and other defense lines (e.g. using IgA, unless the patient dies before this can occur). But when the immune system is overwhelmed by the combined effects of too much oxidative/nitrosative stress and malnutrition, this may have the unfortunate consequence that the remaining defense lines will fall as well – with the result that the patient will die.

There are several different pathogens, including viruses [88–96], [96], bacteria [97–101] and other cellular pathogens [102–104], that are using immunosuppression as part of their offensive strategies (e.g. by inducing the mainly immunosuppressive cytokine TGF-β [98], [99] or by binding to the receptor called DC-SIGN on dendritic cells [100], [101], thus changing the pattern of cytokine expression in favor of the invader and not the host), with the HIV virus being the most well-known (and best studied) example. But it must be a fair assumption that the prognosis in a vast majority of cases will be even worse when some form of immunosuppression caused by the pathogen is combined with immunosuppression being a consequence of malnutrition – which appears to be substantiated by what is known about the importance of nutritional factors not only in connection with HIV disease, but also in various other infections that are commonly associated with (more or less) immunosuppression caused by the pathogen itself, such as measles (for information about immunosuppression by the pathogen in this disease, see refs 88–91,94–96) and tuberculosis (for information about immunosuppression by the pathogen in this disease, see refs 98–101).

In this regard, there are a large number of different nasty pathogens that contain molecules functioning as ligands of the dendritic cell (DC) receptor called DC-SIGN or related proteins. It may be possible that this is a mechanism of immunosuppression that has been exploited by several different pathogens, including viruses, bacteria, and eukaryotic species. However, it needs to be studied in each case whether the DC-SIGN (or related protein) ligand concerned really is immunosuppressive. Binding to the receptor may not necessarily be immunosuppressive per se; it may be possible that immunosuppression will only occur when the ligand from the pathogen functions as an antagonist (blocker) in relation to some physiological ligand that normally has a stimulating and not immunosuppressive effect. If this is so, it may be possible that DC-SIGN ligands coming from various pathogenic species may have different effects on signal cascades downstream of the receptor, i.e. the balance between agonist and antagonist properties in relation to the signal system(s) downstream of the receptor might be different from one case to another. But it may also be possible that there has been an evolutionary exploitation of this opportunity for weakening of the anti-pathogenic defense system of the host in several different lineages, almost regardless of where in the great system of natural taxonomy the pathogenic species concerned might belong. Or to state it in another way: it may be possible that there has been a great deal of parallel or convergent evolution, among several different groups of pathogenic organisms and also some viruses (including HIV-1), which all has led to a similar result.

It may also be possible that immunosuppression resulting from this mechanism may be one of the most important reasons why so many of the pathogens and diseases concerned (including tuberculosis, HIV disease, Dengue fever, SARS, West Nile virus, Sindbis virus, Ebolavirus, hepatitis C, measles, cytomegalovirus, malaria, leishmaniasis, and schistosomiasis) are really nasty and difficult to treat (for documentation I refer to PubMed, where it can easily be found by combining the searchword ‘DC-SIGN’ with the pathogen concerned). From a theoretical point of view, it might be speculated that one of the best therapeutic strategies for countering this form of immunosuppression would be to try to counterbalance the immunosuppressive effect of pathogen-derived DC-SIGN ligands by supplying ligands of receptors that have an opposite regulatory effect on the DCs (e.g. some Toll-like receptor or Dectin-1 ligand), so that a more normal pattern of cytokine secretion from the DCs (with a more normal Th1/Th2 balance) may be restored. It may also be possible that immunostimulating hormones such as androstenediol (AED) in many cases might be useful. This is supported by studies in animals experimentally infected with some of the pathogens concerned, e.g. West Nile virus, showing significant reduction of lethality when using AED (as will be discussed in more detail in a later section of this article). At the same time, it should be considered imperative to correct any specific form of malnutrition that may lead to a skewing of the Th1/Th2 balance in favor of Th2.

There is more than one reason for pathogen-induced immunosuppression, one being that the immunosuppressive pathogenic organisms frequently will alter the balance between immunostimulatory and immunosuppressive cytokines or some of their effects (by changing the expression of a cytokine receptor and thus changing the sensibility of the cell to a given concentration of the cytokine in the extracellular environment) in the same direction as also can happen as a direct consequence of poor nutritional status. Tubercle bacilli, to mention one example, contain two different substances that have been reported to up-regulate the expression of the mainly immunosuppressive cytokine TGF-β [99], [100], but the same can also happen as a consequence of selenium deficiency [105] and/or too much lipid peroxidation – which in turn can be enhanced not only because of selenium or vitamin E depletion (or because of excess intake of polyunsaturated fatty acids), but also because of glutathione depletion – since TGF-β expression is up-regulated by 4-hydroxynonenal [106], which is an aldehyde formed as a secondary product of polyunsaturated lipid peroxidation. Measles virus, to take another example, can alter the Th1/Th2 cytokine balance because the secretion of Th2 cytokines such as IL-10 is up-regulated [90], while the secretion of Th1 cytokines such as IL-12 [94], [96], [107] and IFN-γ [91] can be down-regulated. But the same can also happen as a consequence of glutathione depletion in leukocytes [108–110], while selenium deficiency causes NK (natural killer) cells and T lymphocytes to become less sensitive to growth stimulation by the Th1-associated cytokine IL-2 because the expression of intermediate affinity and/or high affinity IL-2 receptors on NK cells and cytotoxic T lymphocytes goes down [111–114].

It can, moreover, also be theoretically expected that there will be a synergistic interaction between (i) depression of cellular growth rates (affecting leukocytes or their progenitors) as a direct consequence of nutritional deficiency conditions (e.g. a combination of GSH and selenium deficiency causing reduction of the rate of DNA synthesis in leukocytes); (ii) the effects of immunosuppressive cytokines (such as TGF-β or IL-10), the expression of which is up-regulated not only because of poor nutrition, but also because of one or more substances found in some pathogenic organism; (iii) pathogen-induced and malnutrition-induced depression of the secretion of Th1-associated immune-stimulating cytokines such as IL-12, IL-2, and IFN-γ; and (iv) malnutrition-related depression of the sensibility of leukocytes such as NK cells and cytotoxic T lymphocytes to the growth-stimulating effect of IL-2 because the number of high affinity and/or intermediate affinity IL-2 receptors on these cells goes down.

In an earlier paper [6] we explained how malnutrition also can greatly enhance the vulnerability of host cells, tissues and organs to ‘friendly fire’ from the leukocytes. Malnutrition can directly lead to enhancement of the inflammatory response because depletion of antioxidant nutrients such as selenium and GSH will lead to enhanced oxidative activation [6], [115–117] of transcription factors such as NF-κB and AP-1, which in turn causes up-regulation of several different proinflammatory genes, including the genes for TNF-α [6] and IL-6 (for reasons which will be explained below). It can also enhance the rate of intracellular ROS generation for a given concentration of TNF-α (or other cytokines that cause activation of sphingomyelinases) in the extracellular environment [6] at the same time as the cellular capacity for ROS scavenging will be reduced – with depletion of intracellular GSH and of selenium-dependent enzymes here interacting with each other in a synergistic, i.e. multiplicative fashion [6]. At a tissue and organ level, malnutrition can also increase host vulnerability because it may lead to imbalance between endothelial cell production of the vasodilator nitric oxide (NO) and its antagonist superoxide anion radical at the same time as production of another critically important vasodilatory substance, i.e. prostacyclin, also is inhibited [6]. When two of the most important vasodilatory signal substances simultaneously are taken out (at least to some extent), while the production of vasoconstrictor substances continues more or less as before (if it is not even enhanced), the consequence can easily be some kind of local circulatory failure which may lead to the death of the tissue or organ concerned. It may be added that prostacyclin and NO also have important anti-aggregating effects; another likely consequence of too much decreased production of these substances is therefore enhanced tendency for thrombosis (which presumably might range, depending on the severity of the functional disturbance, from scattered microthrombi to massive disseminated intravascular coagulation).

But the malnutrition-induced enhancement of host tissue vulnerability (because of enhanced inflammation combined with decreased resistance to its deleterious actions at a cellular and tissue/organ level) will probably not contribute in any important way to the fight against the pathogenic invader. The consequences might therefore be compared to some factor (e.g. malnutrition, some genetic disturbance or interaction with another drug) that will enhance the likelihood of serious negative side effects of a drug, but without helping in any way to enhance its therapeutic effect. The ‘therapeutic ratio’ for the immune system can thus be drastically decreased when malnutrition causes a drastic blunting of its therapeutic efficacy (i.e. its capacity for killing the pathogenic invader or stopping its replication) at the same time as the negative side effects of the immune system's operations are also drastically enhanced.

The macaque experiments showed that IL-6 hypersecretion most likely may play a very important role as a causative factor of organ damage and death when this primate species is infected by hypervirulent influenza [35]. In humans, it may be possible (and most probable) that not only IL-6, but also other cytokines including TNF-α may be important as contributory causes of the fatal outcome of a cytokine storm. While the role of nutritional factors as important co-determinants of organ vulnerability to the harmful effects of TNF-α overproduction has been reviewed in an earlier article [6], and there should be no need to repeat this here, some words might be added about the way nutritional factors may influence both the rate of IL-6 production and the downstream sensitivity of target cells to its actions.

Nutritional factors may affect the rate of IL-6 production and secretion for basically the same reason as they do for TNF-α (and also several other cytokines), i.e. because expression of the IL-6 gene is under partial control by transcription factors such as NF-κB [118–121] and AP-1 [119], [120] which themselves are redox-regulated [115–117].

Downstream effects of IL-6 are most likely also very sensitive to the nutritional status of the host cells, but for quite other reasons than for TNF-α. Some important mechanisms by which nutritional factors may interfere with signaling downstream of TNF-α have been explained in an earlier survey article [6]; we shall now see how nutritional status theoretically also must be expected to have a strong effect on signaling downstream of IL-6.

IL-6 belongs to a family of 10 cytokines, which all act via receptor complexes containing the cytokine receptor subunit gp130 [122]. Other members of this family include IL-11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1 [123]. These so-called IL-6-type cytokines play an important role in the regulation of complex cellular processes such as gene activation, proliferation, and differentiation [123]. On cells, IL-6 first binds to a specific membrane-bound IL-6R and the complex of IL-6 and IL-6R interacts with gp130 leading to signal initiation [122]. Whereas gp130 is widely expressed throughout the body, the IL-6R is only found on some cells including hepatocytes and some leukocytes [122]. A soluble form of the IL-6R, however, is an agonist capable of transmitting signals through interaction with the gp130 protein [122]. Signaling downstream of gp130 involves the Janus kinases (tyrosine kinases) Jak1, Jak2, and Tyk2, the signal transducers and activators of transcription STAT1 and STAT3, and the tyrosine phosphatase SHP2 [123], while termination of signaling downstream of IL-6, IL-6R, and gp130 happens by different processes involving tyrosine phosphatases, proteasome, Jak kinase inhibitors SOCS (suppressor of cytokine signaling), protein inhibitors of activated STATs (PIAS), and internalization of the cytokine receptors via gp130 [123]. Although all IL-6-type cytokines signal through the gp130/Jak/STAT pathway, comparison of their physiological properties shows that they elicit not only similar, but also distinct, biological responses [123].

The most important (but not the only) reason why nutritional status may affect signaling downstream of the IL-6/IL-6R complex is the inhibition of various tyrosine phosphatases caused by oxidative stress [124–126]. Tyrosine phosphatases reverse the effect of tyrosine kinases, such as those being activated by IL-6. It follows as a consequence that much oxidative stress (e.g. ROS from activated leukocytes) or a weakening of the intracellular system of antioxidative defense (e.g. impaired scavenging of H₂O₂ because of selenium deficiency or GSH depletion, as explained in our earlier review article [6]) may lead to amplification of intracellular signaling downstream of IL-6/IL-6R/gp130 because of reduced activity of protein phosphatases. Oxidant stress-induced enhancement of IL-6 expression and oxidant stress-induced amplification of IL-6 downstream signaling must, of course, be expected to interact with each other in a synergistic (multiplicative) fashion, and it must also be expected that nutritional deficiency conditions leading to impairment of the intracellular system of antioxidative defence (e.g. GSH depletion or selenium deficiency) here will produce some kind of second-order effect. Since IL-6 also can stimulate C-fibres [57], and since C-fiber products such as substance P can make an important contribution to the activation of leukocytes such as macrophages, mast cells, and T cells [127–129], it is not difficult to see how the consequence of immune system overstimulation (by far more viruses than will be produced in a case of normal influenza) combined with poor nutrition status could lead to activation of a vicious circle mechanism, which will lead to amplification of the inflammatory process in the lungs and as a side effect may cause development of an alveolar edema that ultimately might kill the patient.

Conjunctivitis as a low-risk mechanism of natural immunization

There have been numerous outbreaks of highly pathogenic avian influenza in domestic bird populations (different HN subtypes) over the last 50 years [14], and some of these outbreaks (such as the H7N7 oubreak in the Netherlands in 2003) have caused infection of several humans who have been in contact with the animals, but in most cases only as a mild disease mainly in form of conjunctivitis, even though there have also been some few fatal cases [14], [130]. The present H5N1 epizootic seems to be different from most other outbreaks of highly pathogenic avian influenza in the sense that the total reported number of fatal human cases is much higher than in all other outbreaks of highly pathogenic avian influenza that have been reported over the last 50 years. But this perhaps might be explained mainly as a consequence of much larger geographic dispersal of the highly pathogenic viruses this time, while the other outbreaks mostly have affected much smaller areas and possibly may not have been associated with any significant transfer of highly pathogenic viral strains from domestic bird populations to wild birds.

We might now try to turn the question the other way: why have there been so many mild human cases and so few fatal cases associated with earlier outbreaks of highly pathogenic forms of avian influenza in domestic bird populations? Is it because the virus per se is not highly pathogenic for humans, but only for birds, or is there some other possible reason?

This question may now have received a plausible answer with the discovery that the ‘avian’ type of influenza virus hemagglutinin receptor, i.e. the α-2,3 type of receptor, is found in humans not only in the lower airways (in the alveoles and terminal bronchioles), but also in the conjunctiva [14]. The conjunctiva, however, is a tissue separated from the lungs by tissues containing mainly α-2,6-receptors on the surface of infectable cells, while the mucus contains many virus-binding molecules (mucins) that contain α-2,3-linked sialic acid [14].

It might thus be speculated that the eyes may function as organs of natural immunization, where it is possible for avian-adapted (α-2,3-adapted) influenza viruses to cause a local disease that is enough to produce good mucosal immunization (with development of good IgA immunity that subsequently also can protect other organs such as the lungs), but without any serious risk of causing fatal lung disease. The eyes will easily be infected, of course, simply by rubbing them with a dirty finger, which not only children, but also adults easily can do almost without noticing it themselves. But it is not always a bad thing to become infected, when it can happen in such way as to provide good immune protection against a potentially highly dangerous pathogen, but without causing any serious disease. It might be speculated that the same mechanism might explain many of those cases of H5N1 seropositivity which have been found [2], [131] among people who have been in contact with birds harboring highly pathogenic H5N1 avian influenza, but without developing serious pulmonary disease.

It may be important that such cases of H5N1 seropositivity in humans without serious disease (even though there might be milder systemic influenza-like symptoms mainly as a consequence of cytokine release from the infected tissues) should not be interpreted to mean that the virus is not so dangerous for humans, after all. If the virus hemagglutinin should happen to change its receptor-binding properties so that it will prefer binding to the α-2,6- rather than the α-2,3-type of receptor, we must expect that it will no longer be able to cause local infection only in the eyes without spreading to all of the respiratory tract. This could possibly mean that it may be the case fatality among registered and confirmed cases that gives us a more correct picture of the virulence properties of the virus once it is able to reach the lungs, and not the ratio between deaths among the confirmed cases until now and the total number of H5N1 seropositive individuals in the same population, which in most cases may have arisen as a consequence of local infection in the eyes. It may thus be possible that the main consequence of counting human cases of H5N1 seropositivity in the general population (rather than counting the number of deaths among confirmed cases) easily could be to give us a feeling of false security, while disguising the true nature of a still highly deadly threat.

New treatment modalities?

In our earlier review article [6], we not only recommended immunonutrition (i.e. ensuring that the immune system has adequate supplies of all nutrients needed for its optimal function), but also antioxidant therapy (to reduce harm caused by reactive oxygen and nitrogen species that may be produced as a direct consequence of the ‘cytokine storm’). We also recommended endothelial protection therapy (to prevent tissue or organ death as a consequence of dysregulation of the local circulation), and the use of melatonin at a high dosage level, not only because of its function as an important immunomodulatory agent, but also because it has powerful antioxidative protective effects and helps to up-regulate the expression of antioxidant enzymes.

There seems to be no need to revise any of these recommendations in the light of the new research data that have appeared– even though it should be re-emphasized that it is not enough here to extrapolate from what has been found in studies with other diseases either in experimental animals or human patients. There is also a desperate need to test the suggested new treatment methods not only in animals experimentally infected with lethal influenza viruses (which to our knowledge not has been done so far), but also more directly in spontaneously occurring human cases of hypervirulent H5N1 influenza (with testing of the suggested new treatment methods in experimentally infected human volunteers being impossible for ethical reasons). We did not specifically discuss possible advantages or shortcomings of the new suggested therapeutic strategy compared to the treatment methods (with anti-influenza drugs) that are used today. But there is no obvious reason to believe that the two treatment methods should interfere with each other in any harmful way. It is more likely that a synergistic interaction might occur, so that the drugs now being recommended hopefully might also give substantially better effects when used as part of a multifactorial and well-integrated treatment strategy (which incorporates all the other treatment modalities mentioned above) than when being used as monotherapy. At the same time, it is imperative when trying to find more efficacious multifactorial treatment strategies, to try to find ways of doing this as simply, safely, and cheaply as possible, so that it can be done not only in the best and most well-equipped university hospitals of the affluent countries, but even in the situation of a poor African village where the nearest medical doctor is 200 km away (or in the graveyard, having himself succumbed to the same virus).

In addition, there might be other possible treatment modalities (not mentioned in our article), which on the background of what is known today would seem to deserve closer attention – either for immediate clinical testing or for inclusion in an international research program with the aim of finding much better treatment methods for this very serious disease as soon as possible. Since we cannot predict exactly when a new pandemic will come, but should not disregard the possibility that it might happen very soon, priority should be given to making use of relevant knowledge already available from other fields of medical research (e.g. from treatment of cancer), rather than finding drugs that are entirely new and possibly might require several years to find and develop.

We should perhaps recall the history of what happened in England during the late 1930s, when a team of scientists led by Howard Florey set themselves the task of trying to scan available scientific literature for information that possibly might be used for developing new and more effective methods for treatment of bacterial infections, and to pursue those observations (through more experimental work) that appeared most promising [132]. They first opted for lysozyme, which was not a success. But when they understood that this substance did not work as they had hoped, they turned instead to the next item on their list, based on a little recognized and more or less forgotten laboratory observation from 1928 – i.e. penicillin [132]. This substance looked far more promising, but the most difficult task was still ahead, which was to mobilize those economic, technical, and scientific resources that were needed to develop methods to produce it cheaply enough on an industrial scale. This task was also solved, but only thanks to an unprecedented technical development program where several different American pharmaceutical companies collaborated with each other, setting normal competition and commercial considerations aside in a common effort to try to save the lives of American and allied soldiers in the battlefields of the Pacific and Europe [132].

It should also be emphasized that the most serious challenge now is to find treatment methods that can be used not only in well-equipped hospitals in North America and Europe, but also could be safely used, and on a mass scale, in those poor countries where there is reason to expect that the death toll in the absence of effective immunization will be highest. Many of these countries have already now, in a pre-pandemic situation, far too few qualified doctors and nurses, and it must be expected that the capacity of their health systems will be absolutely overwhelmed, even more than the health systems in the rich countries, if there should be a new pandemic with something that has a lethality (for untreated patients) similar to what happened during the Black Death. What we need for these countries is therefore not only something that can be produced very cheaply (and at the sufficient scale within a short time), but can also be used safely by non-professionals and by traditional healers. Priority should therefore also be given to try to find such treatment modalities or drugs that are associated with a minimum of toxicity and risk of serious side effects, even when being used in the hands of people who are not only medical non-professionals, but some of them even with limited literacy skills.

A good strategy of defense should ideally be based on a best possible understanding of what the enemy is doing, or what he plans to do, both as regards the nature and quality of his weapons and his overall strategy. What we are dealing with in this case is an enemy who attacks by first of all using crushing force to take out all the radar installations and associated anti-aircraft and anti-missile weapon systems of the country he is going to invade. It is the neutralization of an important intracellular system for detection of RNA viruses and the associated antiviral weapon systems (being part of the system of innate immune responses) that probably should be regarded as the basic key for a better understanding of the pathogenesis of this dreadful disease. The neutralization (or immobilization) of this viral recognition and antiviral defense system also means that a very important system of negative feedback control of the rate of viral replication no longer is operative. In the case of normal influenza viruses, this system may be able to control the viral load because the strength of the anti-proliferation brake mechanism is increased (at least, if not even more) in direct proportion to the number of virus particles. It means that the number of virus particles can rise up to a certain plateau level, but not higher (analogous to a thermostat controlling a system of electrical heating in a house). However, when the ‘thermostat’ is destroyed, the virus will instead continue to proliferate until the number of virus particles is higher perhaps by a factor of 10 compared with what should have been the plateau (or saturation) level in a case of normal influenza.

What might still be operative are perhaps other virus recognition systems associated with the system of innate immune response (e.g. some of the Toll-like receptors?), and also various adaptive immune response mechanisms. The adaptive immune system is in principle capable of coping with almost any kind of enemy, including this one. The main limitation of this system lies in the long time it requires for efficient response to an enemy which it has never seen before. There is a vast difference between the rate of replication of RNA viruses and the rate of replication of human cells, even more so than when we compare the rate of division of bacteria and the rate of division of human cells.

What should be regarded as the most important task of the general in charge of the defense forces, i.e. the physician, is therefore simply to try to keep the patient alive and with a minimum of permanent organ damage for the time it takes until it is possible for the adaptive immune system to mount an effective counter-attack. It is also important to give the cells of the adaptive immune system all the nutrients they need in order to multiply as fast as possible. They should further be given orders (using suitable immunostimulators, e.g. immunostimulating hormones or some suitable Toll-like receptor agonist) to hasten as much as possible with this task of proliferation. If there is anything that can be done to strengthen those parts of the innate immune system that still might be operative (and which actively can help to limit the advance of the viral invader), this should, of course, also be done.

An exhaustive overview of all methods and substances that might deserve research attention in a broad program for developing a better treatment strategy for hypervirulent influenza will not be attempted here. This is a task which is better left to an international expert group with a mandate similar to the task of the group of British scientists who were responsible for the ‘rediscovery’ of penicillin in the library [132]. But some of those therapeutic principles and substances that ought to be taken into consideration (in addition to what already has been mentioned in our earlier review article) will be mentioned below, although not with the intention of giving an exhaustive overview of what is known about each substance (and that may be relevant in this context). Much of this can easily be found in literature databases such as PubMed by readers when they know what to look for. The intention is more to give the readers some general idea of some of those directions where they might go, if they want to search in textbooks (other than those of their own specialty as a physician or medical scientist), scientific monographs or literature databases for already available scientific knowledge that might be relevant in this particular context. Each new apparently promising approach will, however, require research to determine whether or not it is actually useful.

Immunostimulation

There are at least two important reasons why immunostimulation must be considered very important. One is the severe defect in important innate mechanisms of antiviral defense that now seems to be fundamentally important not only in the pathogenesis of influenza caused by hypervirulent H5N1 viruses, but also in the pathogenesis of Spanish flu influenza. The other reason is the leukopenia and lymphopenia that have been observed in human patients with H5N1 influenza, and that quite certainly cannot be expected to improve their chances of survival when it comes on top of failure (caused by the virus itself) of important innate mechanisms of antiviral defense which are dependent on a normal production of type I IFNs. While good nutrition for the immune system should be considered as the basis for all efforts aimed at maintaining (or restoring) the function of this system in patients suffering from hypervirulent influenza, there may also be various other things that may be done. Much of it, if it can be demonstrated to work, can presumably also be done very cheaply and with so little risk of serious harmful side effects that it would not be problematic to leave responsibility for the therapy to paramedical personnel – in a situation where the number of seriously ill patients is so large compared with the number of qualified health personnel that it will be impossible for the latter to reach more than a minuscule proportion of the patients needing medical attention and care.

Melatonin may be of interest here for various reasons [6], not least its safety of administration (very low risk of serious side effects) and cheapness of production being important in this particular context. Melatonin has been reported to selectively counteract bone marrow toxicity when administered together with cancer chemotherapeutic compounds, but without interfering with their anticancer action [133]. In vitro, melatonin was shown to counteract apoptosis in bone marrow cells incubated with etoposide [133]. Such protection was reflected by an increased frequency of granulocyte/macrophage-colony forming units, but not of the pluripotent spleen-colony forming units [133]. The effect of melatonin was found to be neutralized by anti-granulocyte/macrophage-colony-stimulating factor monoclonal antibodies (mAbs) [133]. When athymic, T-cell-deficient mice were used as bone marrow donors, melatonin did not exert any protective effect [133]. It was therefore thought that melatonin might be able to stimulate the endogenous production of granulocyte/macrophage-colony-stimulating factor via bone marrow T cells [133].

Later, it was reported that the colony-stimulating activity of melatonin as well as its ability to protect myeloid progenitors from apoptosis induced by anti-cancer drugs are mediated mainly by T-helper cells producing opioid cytokines [134]. These putative new cytokines have been named melatonin-induced opioid (MIO) [134]. The hematopoietic effect of melatonin has been reported to involve type 1 kappa-opioid receptors on bone marrow macrophages and possibly interleukin-1 [135]. Melatonin, moreover, has also been reported to counteract thrombocytopenia [136–138], which may presumably reflect much of the same type of mechanisms. There should thus be good reason for hope that melatonin might help to boost the effect (prophylactically or therapeutically) that good nutrition hopefully could have in relation to the problem of leukopenia in patients suffering from hypervirulent H5N1 influenza.

DHEA and some of its hydroxylated metabolites have been reported to have important immunostimulatory and immunomodulatory effects [139–147]. The effects of these substances are dramatically different in vitro, with androstenetriol being far more potent than the others [143], but more similar in vivo owing to metabolic conversion first of DHEA to androstenediol and next of androstenediol to androstenetriol [143]. DHEA is reported to function as an anti-glucocorticoid hormone counteracting the immunosuppressive effect of glucocorticoids [141], [147]. It is possible that this could be important in H5N1 influenza because a ‘cytokine storm’ [2] must be expected to lead to strong stimulation of C-fibers in the airways by IL-6 [57] and TNF-α [54–56], and other inflammatory mediators will also be produced (some of them probably in great quantity) that will have effects going in the same direction. It is known from animal experiments that C-fiber stimulation is associated with a central nervous reflex causing stimulation of the secretion of ACTH from the pituitary gland [148–150]. DHEA has also been reported to have neuroprotective and antioxidative protective effects, perhaps mainly mediated by regulatory effects on the expression of enzymes participating in the antioxidant defense system of the cells [151–154]. Moreover, it has been reported to inhibit the respiratory burst in macrophages [139]. Androstenetriol has also been reported to improve survival significantly when administered subcutaneously in a single dose in a rodent model of traumatic shock [155].

It is thus possible that the protective effect of this substance in serious infectious disease, as observed in animal experiments [142], [156–164], may arise because of the combination of direct immunostimulatory effects (perhaps caused mainly by androstenetriol), an anti-glucocorticoid effect, and improved cellular antioxidative defense (which may help various vulnerable tissues and organs to cope better with the strongly enhanced oxidative and nitrosative stress associated with serious infection). This is similar to the well-documented double protective effect (both as an immunostimulant and an antioxidative protector) that has been also found for melatonin, and it is not unreasonable that melatonin and DHEA might interact with each other in an additive or synergistic fashion, if they are administered together. However, this question is something that needs study (the sooner the better!) in animal experiments, using viruses or bacteria that are highly lethal to the animals. It should be noted that it may not necessarily be the same substances (DHEA itself or some of its metabolites) that are responsible for all of the protective effects that have been observed with DHEA. For the time being, it may thus be possible that it would be more prudent to use DHEA itself as a drug for H5N1 patients rather than one of its much more active metabolites, such as androstenetriol. This question, however, is also something that deserves closer study in animal experiments.

Some of the observations done on hematologic and immunologic effects of androstenetriol (AET) and androstenediol (AED) would appear to be of relevance in connection with the problem of leukopenia and lymphopenia that has been observed in human patients suffering from H5N1 influenza [2]. These substances, as already mentioned, have been reported to up-regulate host immunity, leading to increased resistance against infections [145]. AET augments IL-2, IL-3, and IFN-γ levels, and counteracts hydrocortisone immune suppression [145]. AET and AED at a dose of 0.75 mg and 8.0 mg per 25 g mouse, were found to protect 60% and 70%, respectively, of C57/BL/6J mice irradiated with a lethal dose [145]. These hormones also protected mice irradiated with 6 Gy and infected with a Coxsackievirus B4 LD50 [145]. AET significantly increased spleen lymphocyte numbers at 7, 14, and 21 days after a 6-Gy exposure [145]. Fluorescent activated cell-sorter analysis of irradiated mice, spleen, and bone marrow showed that AET significantly augmented the myeloid precursor markers, CD11b/Mac-1 and B220 (pan B), as well as the absolute numbers of CD4⁺/CD8⁺ cells over the 21 days of testing [145]. Overall, these data were thought to be consistent with AET/AED inducing a more rapid recovery of all hematopoietic precursors from the small number of surviving stem cells [145]. AED, moreover, has also been reported to enhance survival after high doses of whole-body ionizing radiation in mice [146]. It was found to stimulate myelopoiesis, to ameliorate neutropenia and thrombocytopenia, and to enhance resistance to infection after exposure of animals to ionizing radiation [146].

The effects observed with AED in relation to myelopoiesis, neutropenia, and thrombocytopenia in experimental animals are obviously of great interest, as seen in relation to the problem of leukopenia in human patients with H5N1 influenza. They resemble those effects on myelopoiesis, neutropenia, and thrombocytopenia that have been observed with melatonin, and it is not unreasonable to consider that AED and melatonin here might interact with each other in synergistic fashion (so that the best therapy for leukopenia in H5N1 patients possibly might be a combination both of immunonutrition, melatonin, and AED). The observations of effects on AED and AET on levels of cytokines such as IL-2, IL-3, and IFN-γ would likewise appear highly relevant in relation to the problem of lymphopenia also observed in H5N1 patients [2]. It should be noted that we are here dealing with substances that probably can be produced rather cheaply (calculating the cost of production per dose needed per patient per day) and also so relatively non-toxic that it would probably be possible to use them safely even in situations where there are extremely few qualified doctors and nurses as compared with the number of seriously ill patients.

We may now turn to various other studies that have been carried out with these substances in experimentally infected animals, mostly with highly dangerous pathogens associated with high lethality in the species concerned. DHEA has been reported to have a significant protective effect in mice infected with West Nile virus, Sindbis virus neurovirulent, and Semliki Forest virus [156]. Mice injected subcutaneously with a single injection of DHEA (1 g/kg) on the same day or 1 day pre- or post-infection with West Nile virus resulted in 40–50% mortality as compared with 100% mortality in control injected mice (p<0.05) [156]. The drug was effective following a single subcutaneous injection or serial intraperitoneal injections (5–20 mg/kg) on days 0, 2, 4, and 6 following virus inoculation [156]. Moreover, DHEA injection not only reduced viremia and death rate, but also significantly delayed the onset of the disease and mortality [156]. The administration of DHEA (serial injections of 10–20 mg/kg with or without a loading dose of 1 g/kg) has also been reported to result in a significant reduction in the mortality rate of mice inoculated with West Nile virus and exposed to cold stress (p<0.05) [157]. Virus levels in the blood and brain of the DHEA-treated mice were significantly lower than in the control groups [157]. DHEA also prevented the involution of lymphoid organs in stressed mice [157].

AED has been reported to protect mice against various lethal infections, including a lethal herpes virus type 2 encephalitis and a lethal systemic Coxsackievirus B4 (CB4) infection [158]. AED was found to be up to 100 times more effective in regulating systemic resistance against lethal infection with CB4 than its precursor DHEA [158]. Compared with DHEA, treatment with AED was found to be markedly superior in protecting mice against virus-induced myocardiopathy, pancreopathy, and mortality [158]. In addition to its protective effect, AED (but not DHEA) induced a 3–4-fold proliferation of the spleen and thymus in virus-infected animals; this effect of AED was only seen above a certain threshold dose [158]. Neither steroid, however, showed any significant direct antiviral effect in vitro; similarly, virus tissue titers in vivo were not affected by the hormones [158]. Additionally, both DHEA and AED were found to protect against a lethal infection with Enterococcus faecalis [158].

In another study of the effects of AED in mice with experimentally induced herpes simplex virus type 1 (HSV-1) encephalitis [159], [160], it was found that subcutaneous administration of 320 mg/kg AED 4 h before viral inoculation enhanced the survival of HSV-1-infected mice, while lower doses (32.0–100.0 mg/kg) were without effect [159]. By 6 days post-infection, there was a significant increase in the expression of the chemokines IP-10, MCP-1, and RANTES and the cytokines IL-6 and IFN-γ in the AED (320 mg/kg)-treated mice compared with vehicle-treated controls [159]. Likewise, there was a corresponding increase in IFN-γ and IL-2, but not IL-12 protein in the trigeminal ganglia of AED-treated mice 6 days post-infection [159]. AED treatment also induced a rise in splenic natural killer activity in a dose- and time-dependent fashion [159]. Collectively, these observations were interpreted to suggest that the protective effect following subcutaneous administration of AED is associated with a selective rise in Th1 cytokines (IL-2 and IFN-γ) as well as of natural killer activity [159]. These observations concerning the effects of AED on levels of Th1 cytokines in experimentally infected animals are obviously also highly relevant in connection with hypervirulent influenza.

AED was also found to enhance the expression of IFN-α mRNA, while it decreased the expression of HSV-1-infected cell polypeptide 27 mRNA in the trigeminal ganglion during the acute (day 6 post-infection) infection of mice [160]. However, there was no change in the viral load from the eye or trigeminal ganglion when comparing the AED-treated with the vehicle-treated mice [160]. Neutralization of antibodies to IFN-α, IFN-β, or IFN-α/β, but not control antibodies, blocked the protective effect following AED exposure, confirming the involvement of type I IFNs in the enhancement of survival in AED-treated mice [160]. Collectively, these results were taken to identify innate immunity as a key component in augmenting the survival of HSV-1-infected mice following AED treatment [160].

These observations would appear highly relevant in relation to the problem of deficient type I IFN response in hypervirulent influenza, as seen in the experiment with macaques infected with Spanish flu virus [35]. It must be highly reasonable to speculate that the problem very likely must be the same in human cases of H5N1 hypervirulent influenza (even though it may be possible that this, strictly speaking, should be regarded as a working hypothesis still in need of confirmation by studying blood samples from naturally occurring cases of human H5N1 infection).

The effects of AED in animals experimentally infected with influenza virus have also been studied [161–163], as well effects of AED-sulfate (AED-S) on the immune response following influenza vaccination in aged animals [165]. AED was found to confer protection against lethal infection with influenza A virus [161], [163]. Pretreatment with 320 mg/kg AED protected male mice from lethal influenza virus infection [161]. In addition, AED was found to enhance antigen-induced trafficking of mononuclear cells into the draining lymph node and augment antigen-specific activation of helper T cells, which are important for control of viral pathogenesis [161]. Furthermore, AED prevented the characteristic increase in serum corticosterone noted during influenza A virus infection [161]. AED was also found to prevent glucocorticoid-mediated suppression of IL-1, TNF-α, and IL-2 secretion [163] and to inhibit glucocorticoid-induced transcription of a glucocorticoid-sensitive reporter gene [163].

In yet another study, the goal was to analyze the ability of AED to counter-regulate the influences of stress on antiviral immune responses [162]. Male C57BL/6 mice treated with 320 mg/kg AED were infected with influenza virus and subjected to repeated cycles of restraint [162]. AED was found to block restraint-mediated suppression of cell recruitment to the draining lymph node, lung NK cell activity, and CD4⁺ T-cell activation [162]. In addition, mice treated with AED had lower pre-corticosterone levels as compared with vehicle controls and the restraint-mediated elevation of corticosterone was significantly blunted by AED treatment [162]. The protective activity of AED in experimental infection with highly pathogenic (for the animals) influenza virus appears therefore to be mediated, at least in part, by suppressing and counterbalancing the immunosuppressive function of regulatory glucocorticoids [163].

In yet another experiment, carried out to examine whether AED could effectively reverse the age-associated decline of antiviral immunity, 3-, 10-, and 22-month-old mice were treated with AED-S for 45 days beginning 10 days before vaccination [165]. Subsequently, mice were primed and boosted with suboptimal doses of a commercially available trivalent influenza vaccine [165]. Treatment of 10-month-old animals with AED-S during vaccination was found to increase the titer of circulating antiviral IgG to levels comparable with those in 3-month-old mice [165]. Furthermore, AED-S treatment protected 10-month-old animals from intranasal challenge with a lethal dose of influenza virus 21 days after secondary vaccination [165]. However, AED-S treatment did not enhance vaccine responses and failed to protect against lethal challenge in 22-month-old mice [165].

The protective effects of AED and DHEA on the pathophysiology of two lethal bacterial infections and endotoxin shock have also been studied [164]. The infections included a gram-positive organism (Enterococcus faecalis) and a gram-negative organism (Pseudomonas aeruginosa) [164]. Both hormones were found to protect mice from lethal infections with these bacteria and also from lipopolysaccharide (LPS) challenge [164]. Treatment of animals lethally infected with P. aeruginosa with DHEA resulted in a 43% protection, whereas treatment with AED gave a 67% protection [164]. Both hormones were also found to completely protect animals infected with an LD50 dose of E. faecalis [164]. Similarly, the 88% mortality rate seen in LPS challenge was reduced to 17% and 8.5%, by treatment with DHEA and AED, respectively [164]. The protective influences of both steroids were shown not to be directly antibacterial, but primarily an indirect antitoxin reaction [164]. DHEA appeared to mediate its protective effect by a mechanism that blocks the toxin-induced production of pathophysiological levels of TNF-α and IL-1 [164]. AED usually had greater protective effects than DHEA; however, the AED effect was independent of TNF-α suppression, both in vivo and in vitro [164].

These observations, especially as regards the effects on toxin-induced TNF-α and IL-1 production, are of great potential interest as seen in relation to the problem of ‘cytokine storm’ in human patients with hypervirulent H5N1 influenza [2]. They agree with an earlier report that DHEA can protect mice from endotoxin (LPS) toxicity while reducing TNF-α production [166].

Effects of DHEA and AED have also been studied in mice experimentally infected with M. tuberculosis [142]. Experimental tuberculosis follows a course in an established model in BALB/c mice in which there is first an early phase of Th1-mediated response accompanied by adrenal hyperplasia, and next a phase where there is a switch to Th2-mediated response, progressive loss of TNF-α expression, and disease progression [142]. Both compounds were found to be protective, particularly AED, which caused a fall in bacterial counts and prolonged survival [142]. These effects correlated with the appearance within 3 days of cellular infiltrates rich in cells expressing IL-2, IL-1α, and TNF-α, and with partial suppression of the switch to IL-4-producing cells that occurred in controls [142]. AED also caused enhanced development of granulomas at 14 days, and persistence of granuloma formation to 120 days, with a corresponding suppression of areas affected by pneumonia [142]. Much of the therapeutic effect of AED and DHEA was obtained by treating for only the first 3 weeks, which is the phase of adrenal hyperplasia [142]. The latter observation was thought to suggest that the ratio of glucocorticoid to anti-glucocorticoid steroids may play a role in the pathogenesis of tuberculosis [142]; it may thus be possible that much of the therapeutic effect in this animal model may be related to the anti-glucocorticoid actions of DHEA and AED, which may also be of relevance in connection with hypervirulent influenza. It might be speculated that the effect on TNF-α production, being opposite to what has been seen in some other studies [164], [166], also might be explained mainly as a consequence of the anti-glucocorticoid action of these compounds.

Thymic hormones can very likely be used to obtain a more rapid immunological response following the start of infection with hypervirulent influenza. It may be possible [7] that changes in the level of thymic hormones as a function of age could have been largely responsible for the steep enhancement of mortality as a function of age that could be observed during the Spanish flu pandemic, comparing children in the age group 10–14 years with young adults in the age group 25–29 years [5]. One reason for the higher production of thymic hormones in children than in young adults is the stimulatory effect of growth hormone on the thymus [167–171]. Prolactin also has a similar stimulatory effect [169], [172], which possibly might help to explain why women of fertile age had a lower mortality during the Spanish flu pandemic compared with men of the same age [5]. But the effect of thymic hormones needs to be tested in animal models of highly virulent influenza (or other viral infections highly lethal for the animals) before it can be tried on human patients. While there should be no serious practical problem associated with using these peptide hormones in a more normal hospital setting in an industrial country, it is not easy to see how it could be safely done in a developing country in a situation where the number of well-educated health personnel is far too small, even in a normal pre-pandemic situation, compared with the number of patients needing treatment. It is also possible that price would be a much greater problem – for mass therapy of a very large number of patients in a poor country – with thymic hormones or their active synthetic peptide analogs compared with substances like melatonin and DHEA (or AED).

β-Glucans are of interest for a number of reasons. Even more than in the case of the immunostimulating hormones melatonin, DHEA, and AED, they are substances that can be produced in great quantities very cheaply, and they appear to be associated with very little toxicity or negative side effects. In this respect, they may thus be considered quite ideal for mass therapy of poor patients in poor countries in a severe medical emergency situation (which might also be considered relevant in connection with the pandemic of HIV disease now affecting large numbers of patients in several poor countries). β-Glucans are found naturally as components of the cell wall in fungi, including many edible species [173–176], but they can also be found in some plants, including oat grains [177]. Mushrooms containing β-glucans (but often also other bioactive substances) have been collected and consumed in China, Korea, and Japan for centuries; for example, Ganoderma lucidum (Reishi), Lentinus edodes (Shiitake), Grifola frondosa (Maitake), Hericium erinaceum (Yamabushitake), and Inonotus obliquus (Chaga) [178]. Having been used for hundreds if not thousands (or ten thousands?) of years in the traditional medicine of some of these countries, their mechanisms of action have now been revealed and their clinical efficacy confirmed by modern scientific studies, much of this research taking place in countries such as Japan and China, but more recently also in Europe and North America.

The biological effects of β-glucans may in an evolutionary sense have arisen as a consequence of the need of early animals (or perhaps their even more ancient pre-animal ancestors) to defend themselves against fungal pathogens (or against pathogens belonging to other groups of early eukaryotes or bacteria that were using β-glucans as part of their cell walls). They are therefore recognized by various cellular receptors, being part of the innate system of immune defense, similar to what happens with molecules characteristic of other broad classes of pathogenic organisms, such as bacterial and viral CpG-rich DNA, double-stranded viral RNA, bacterial flagellin, and various cell wall components typical of large groups of bacteria. β-Glucans are recognized by the Dectin-1 receptor [179–183] and possibly by Toll-like receptors 2 and 6, since these receptors are stimulated by zymosan particles [184], [185] that are rich in β-glucans [186], but are also rich in other structures [186] that possibly might be responsible for activation of the Toll-like receptors 2 and 6, rather than the β-glucans. β-Glucans, moreover, are also recognized by a β-glucan-binding site which is a lectin domain on complement receptor 3 [187–194]. Dectin-1 is not considered as belonging to the group of Toll-like receptors; instead it represents the prototype of innate non-Toll-like receptors containing immunoreceptor tyrosine-based activation motifs (ITAMs) related to those of adaptive antigen receptors [182]. A protein called Card9 has been identified as a key transducer of Dectin-1 signalling [182]. It controls Dectin-1-mediated myeloid cell activation, cytokine production and innate antifungal immunity [182].

Complement receptor 3 (CR3, also known as Mac-1, CD11b/CD18, or αMβ₂-integrin) functions as an adhesion molecule and a receptor for factor I-cleaved C3b, i.e. iC3b [189]. It is found on various types of leukocytes including neutrophils [188], [193–196], eosinophils [195], [197], macrophages [188], [198–201], DCs [202–204], and natural killer (NK) cells [187], [188]. Pathogens that activate complement are first coated with the C3b fragment of C3, which is rapidly proteolyzed into the iC3b fragment by serum factor I [187]. These iC3b fragments serve to promote the high avidity attachment of the ‘iC3b-opsonized’ pathogens to the iC3b-receptors (CR3, CD11b/CD18) of phagocytic cells and NK cells, stimulating phagocytosis and/or cytotoxic degranulation [187]. When β-glucans bind to the lectin domain of this receptor, they function as costimulators altering signaling downstream of the receptor after it has also bound iC3b [189]. This results in priming of the iC3b receptor for cytotoxicity directed against iC3b-opsonized target cells [189].

A combination of β-glucans and antibodies against tumor-specific antigen has been tested in animals as a new method for immunotherapy of cancer with very promising results [187], [189–192], [194]. The tumor-specific antibodies may help various types of effector cells, including granulocytes [190], to recognize their target, while the β-glucans give orders to kill (so to speak). The combination of β-glucans and tumor-specific antibodies is therefore much more effective for eliminating tumor cells than the tumor-specific antibodies alone. However, it is very difficult to see, if this principle can work for tumor cells, why it should not also work for cells infected with viruses or with intracellular bacteria such as M. tuberculosis (or any eukaryotic pathogen that lives inside some of the cells of its host organism). There may, after all, be very few viruses or bacteria that are capable of deploying such a broad-spectrum arsenal of potent anti-immunological defense weapons as tumor cells. And there should be good reasons for hope that the same principle also might work against some of the multicellular parasites attacking humans or domestic animals.

When a person develops adaptive immunity after a naturally acquired respiratory infection (as distinct from vaccination using intradermal vaccines), IgA-dependent immunity is very important for protecting against new attacks (except perhaps in a very mild form) by the same pathogen. But IgA is capable of binding C3b [205]. So there might be some reason for hope (even if it probably remains to be tested), that the same principle that has been shown to work for cancer cells (at least in animal experiments) could work for almost any kind of infection in the airways, when the right type of anti-pathogenic IgA can collaborate with exogenously applied β-glucans.

The Dectin-1 receptor can be found on neutrophils [206–208], eosinophils [207], monocytes [207], macrophages [206], [207], DCs [206], [207], B-cells [207], and a subpopulation of T cells [207], but its expression on a given cell type can, at least in the mouse, be very different, comparing different anatomical locations [206]. The expression of the Dectin-1 receptor on murine leukocytes in situ correlates with its function in pathogen recognition and reveals potential roles in leukocyte interactions [206]. The Dectin-1 receptor is also called the β-glucans receptor (βGR) [207]. It has two different major isoforms, βGR-A and βGR-B [207]. Both of these isoforms are expressed by cells in peripheral human blood [207]; however, only βGR-B is significantly expressed on mature human monocyte-derived macrophages and immature dendritic cells [207]. It has been shown that Dectin-1 acts as the major β-glucan receptor on mature human macrophages [207]. The expression of Dectin-1 on human blood cells can be regulated up or down [209].

The effect of β-glucans binding to Dectin-1 depends on the nature of the β-glucan or perhaps (similar to what is known for immunoglobulins) on the distance between β-glucan motifs binding to different Dectin-1 receptor molecules on the same cell, since not only anti-Dectin-1 antibodies, but also soluble β-glucans were found to inhibit phagocytosis, ROS production, and killing of Candida albicans by polymorphonuclear leukocytes [208].

It might also be speculated, since we are dealing with a typical ‘pattern recognition’ molecule helping not only to recognize pathogenic invaders, but presumably also to distinguish between potentially dangerous pathogenic organisms and harmless commensals, that the effect of β-glucans binding to Dectin-1 might depend strongly on the presence of other molecules functioning either as positive regulators (co-stimulators) or negative regulators of the same cells. It has been shown in connection with studies of the recognition of pathogenic fungi by macrophages and DCs that while Dectin-1 signals alone are sufficient to trigger phagocytosis and Src-Syk-mediated induction of antimicrobial ROS, collaboration with TLR2 signaling enhances NF-κB activation and regulates cytokine production [210]. It might, moreover, be speculated that one of the most important forms of co-regulation of signaling downstream of Dectin-1 possibly might be mediated by β-glucans themselves when binding to another receptor, i.e. the complement receptor 3, depending on whether or not the complement factor fragment iC3b is also present, as well as on the concentration of the latter molecule, especially when it is attached to immunoglobulins.

Dectin-1 ligation by zymosan particles or live C. albicans yeast has been shown to trigger activation of the transcription factor NFAT (nuclear factor of activated T cells) in macrophages and DCs [210]. Dectin-1-triggered NFAT activation plays a role in the induction of early growth response 2 and early growth response 3 transcription factors, and also cyclooxygenase-2 [210]. It has been shown that NFAT activation regulates IL-2, IL-10, and IL-12 p70 production by zymosan-stimulated DCs [210]. Binding of β-glucans to Dectin 1 can also synergize with Toll-like receptor 2 ligands to induce TNF-α and IL-12 [211]. Dectin-1 can promote synthesis of IL-2 and IL-10 through phosphorylation of the membrane proximal tyrosine in the cytoplasmic domain and recruitment of Syk kinase [211]. syk-/- DCs do not make IL-10 or IL-2 upon yeast stimulation, but do still produce IL-12, indicating that the Dectin-1/Syk and Dectin-1/TLR2 pathways can operate independently [211]. Tyrosine kinase-mediated signaling is strongly co-regulated, as mentioned earlier, by the level of oxidative stress inside the cells, since tyrosine phosphatases (which antagonize the tyrosine kinases) are inhibited by oxidative stress [124–126]. Oxidative stress, moreover, can also activate various tyrosine kinases including Syk [212], [213]. It may therefore be expected that oxidative stress will up-regulate the Dectin-1/Syk signal pathway, in turn causing up-regulated expression of IL-10 and IL-2. This might in turn be expected to change the IL-12/IL-10 balance, or Th1/Th2 balance (since IL-12 is a Th1 cytokine, while IL-10 is a Th2 cytokine), in favor of IL-10, which might possibly lead to enhancement of the Th2 response and weakening of the Th1 response. So it is possible that too much production of ROS outside or inside the cell or poor nutritional status as regards nutrients needed for the normal function of ROS-scavenging enzymes (such as selenium, sulfur amino acids, and various B-group vitamins) could lead to a toppling of the Th1/Th2 balance as a consequence of this mechanism, with nutritional status affecting one of the signal pathways downstream of the Dectin-1 receptor more than it affects the other. However, these changes may be opposed by enhanced expression of IL-2, since IL-2 is a Th1 cytokine, and it may be possible that the net outcome may depend on other co-regulatory factors.

It can thus be seen that the Dectin-1 receptor functions as part of a highly sophisticated and versatile regulatory system in the DCs; it does not function only as a simple positive regulator like the gas pedal in a car. Nutritional status and also the total level of oxidative (and nitrosative) stress associated with inflammatory activity in a tissue seem to function as very important co-regulators.

Nutritional status also seems to be important as a co-regulator of signaling downstream of the Dectin-1 receptor in macrophages, as illustrated by a study of the effects of lentinan (which is a β-glucan preparation made from the fungus Lentinus edodes) on peritoneal macrophages [108]. Lentinan reduced the production of prostaglandins, IL-10, and IL-6 in the macrophages, while it enhanced the capability of macrophages to produce IL-12 and nitric oxide (NO) upon in vitro triggering [108]. However, this effect was dependent on an elevated level of intracellular glutathione (GSH) content in the macrophages [108]. Deprivation of intracellular GSH completely ablated the production of IL-12 [108]. Conversely, LPS caused reduction of the intracellular GSH content and a reciprocal profile of mediator production [108]. Macrophages with elevated intracelluar GSH can be arbitrarily designated as reductive macrophages (RMp) and those with reduced amount of GSH as oxidative macrophages (OMp) [108]. OMp were found to be converted to RMp when GSH was replenished with glutathione monoethylester [108]. Administration of IL-2 in combination with lentinan was found to exert a synergistic induction of RMp, which in turn resulted in synergistic augmentation of IL-12 and NO and reduction of IL-6 production [108]. It was also found that CD4⁺ T cells from mice which had received lentinan showed augmented IFN-γ production and reduced IL-4 production upon in vitro anti-CD3 stimulation [108]. It was concluded that skewing of Th1/Th2 balance towards Th1 by a β-[1–3]-glucan, lentinan, is directed through the enhanced production of IL-12 and reduced production of IL-6, IL-10, and prostaglandin E₂ by macrophages, depending on intracellular GSH redox status [108]. If β-glucans are to be used for efficient tumor immunotherapy, it may be one of the critical elements to induce a reductive form of macrophages in tumor stromal tissues, i.e. enhance their intracellular GSH concentration, to maintain the Th1 response [108].

The effect found in this study of the combination of high intracellular GSH concentration and β-glucan administration on the secretion of IL-6 is of special interest in connection with hypervirulent influenza, since IL-6 appears to be one of the most important cytokines associated with the ‘cytokine storm’, if not the most important one, as illustrated by the experiment with Spanish flu virus infection of cynomolgus macaques [35]. It may thus be possible that co-administration of β-glucans and glutathione (or some suitable GSH precursor or GSH/sulfur amino-rich food, e.g. dried whey, whey protein concentrate or fish protein concentrate type B) might be used as a method for diminishing the overproduction of IL-6 in the lungs of patients suffering from hypervirulent influenza. The best route of administration of the β-glucans would in this case possibly be in form of an aerosol spray preparation for inhalation, similar to glucocorticoid preparations for asthma patients. Further, the question might be raised as to what extent the overproduction of IL-6 that was observed in the macaque/Spanish flu experiment [35] may have happened as a direct consequence of intracellular GSH depletion in macrophages and perhaps other leukocytes.

It can be seen that the β-glucans have important regulatory effects not only in relation to the effector functions (such as respiratory burst and degranulation) of leukocytes considered to be part of the innate immune system, but also in relation to the adaptive immune response. The regulatory effects of β-glucans on the adaptive immune system arise in part as a consequence of their regulatory effects on the production of Th1-type versus Th2-type cytokines in DCs and macrophages, but can also be mediated by Dectin-1 receptors present on B cells and some of the T cells. B-Glucans can, moreover, play a most important role as positive regulators when the innate and adaptive parts of the immune system collaborate through cell killing mechanisms whereby immunoglobulins help to mark those target cells that are to be killed (e.g. tumor cells), while cells belonging to the innate immune system (e.g. neutrophil granulocytes) do the actual job of killing.

β-Glucans can, as already mentioned, function both as positive and negative regulators of immunological functions. One possible example of their role as negative regulators can be seen in a recent report about yeast zymosan functioning as an inducer of regulatory antigen-presenting cells and immunological tolerance [214]. And a soluble β-glucan preparation was found to inhibit experimental periodontal disease in Wistar rats [215]. There are also other reports about β-glucans or β-glucan derivatives having anti-inflammatory effects [216–218], i.e. suppression of TNF-α and IL-6 production in leukocytes in mice treated with various bacterial toxins associated with septic shock [216].

But β-glucans can also stimulate microbicidal host defense mechanisms [219–221], as exemplified by a study of the effects of lentinan in animals infected with a lethal dose of influenza virus [219]. Significant protection was conferred by lentinan administered intranasally before lethal influenza virus infection, which could be corroborated by a reduction of the lung virus titers [219]. Since the lung is the target organ of influenza virus infection, the administration of lentinan by the intravenous route was tested [219]. Lentinan was found to confer complete protection against an LD75 challenge dose of virulent influenza virus and significantly prolonged the survival time after an LD100 challenge [219]. The effect on respiratory burst of bronchoalveolar macrophages was investigated by luminol-dependent chemiluminescence in response to stimulation by zymosan [219]. Enhanced chemoluminescence activity was present at an early stage in groups receiving lentinan [219]. Significant NO activity could also be stimulated by culturing bronchoalveolar macrophages in the presence of lentinan [219]. TNF activity could not be detected in lung lavage, but measurable IL-6 was produced already after 6 h in animals administered lentinan alone and in lentinan-pretreated influenza virus-infected mice [219]. Influenza virus alone did not induce measurable IL-6 at 6 h, but high activity was present at later time periods [219].

The effect of β-glucans has also been studied in mice experimentally infected with tuberculosis [220], [221]. When lentinan was administered intraperitoneally before infection (at a dose of 1 mg/kg three times at 2-day intervals), it was found that peritoneal macrophages obtained from animals treated with lentinan were greatly stimulated, as assayed by establishing their number, acid phosphatase activity, H₂O₂ production, and killing ability against M. tuberculosis in vitro [220]. In another experiment using rats instead of mice, the animals were treated intranasally before infection with lentinan at a dose of 1 mg/kg (administered three times at 2-day intervals) [221]. Samples of bronchoalveolar lavage fluid were obtained from rats at different intervals – 3, 24, and 72 h after infection [221]. It was found that lentinan induces high level of alveolar macrophage activation manifested through enhanced bactericidal effect against M. tuberculosis, which correlates with the induction of reactive nitrogen intermediates, increased activity of lysosomal enzymes (acid phosphatase), and with effective phagolysosomal fusion followed by destruction of mycobacteria [221].

β-Glucans can also be used as adjuvants, as has been demonstrated in fish [222–226], in experiments with mammalian species used as laboratory animals [227–231], and in humans [232], [233]. This could presumably be very important for development of new and better vaccines against hypervirulent influenza virus strains – vaccines that might simultaneously be efficacious, cheap, and easy to administer, which are the three main requirements that should be satisfied for a new vaccine that can be used all over the world and not only by the populations of the rich countries. However, it must be reasonable to assume that a similar adjuvant effect may occur during immunization as a consequence of natural infection. Or, stated in a different way, there should be good reason for optimism concerning the possibility of using β-glucans as therapeutic agents to obtain a more rapid and stronger adaptive immune response following the start of infection with hypervirulent influenza.

While vaccines containing β-glucans as adjuvants are currently used in large quantities in veterinary medicine, especially in the form of vaccines for fish, there is apparently no such vaccine commercially available for use either prophylactically or therapeutically (in connection with serious chronic diseases) in humans. But two important feasibility studies have been done [232], [233]. One of these was a comparative study of different candidate adjuvants for use as part of mucosal vaccines to be applied in the vagina [232]. The female genital tract is an important entry site for numerous pathogens, and some of these (especially, of course, HIV disease) are of great public health concern. Local immunization, generating specific mucosal IgA and systemic IgG, could represent an interesting potential opportunity not only for prophylaxis, but also treatment of some of these diseases. If this succeeds, this might be considered an almost ideal therapy method, especially among poor patients in poor countries, where educated health personnel are few compared with the number of patients. However, such a vaccine strategy needs mucosal adjuvants to obtain the best immune response [232]. Considering that the immunization process is mainly dependent on the capture and on the transport of the antigen by Langerhans cells, potential adjuvant molecules were evaluated by analyzing their effects on the CCL20 secretion by endocervical and exocervical/vaginal epithelial cells as well as on DC and Langerhans cell maturation [232]. It was demonstrated that DC-Chol and zymosan (which is rich in β-glucans) were the most efficient mucosal candidate immunoadjuvants that generate a strong increase of CCL20 secretion by the two epithelial cell lines and the maturation of DCs and Langerhans cells, respectively [232].

In the other study, an oral solution of soluble branched yeast β-1,3-D-glucan (SBG) was investigated primarily for assessment of safety and tolerability in an early phase human pharmacological study (phase I) [233]. Eighteen healthy volunteers were included among non-smoking individuals [233]. Groups of six individuals received SBG 100 mg/day, 200 mg/day or 400 mg/day, respectively, for 4 consecutive days [233]. The dose increase was allowed after a careful review of the safety data of the lower dose group [233]. No drug-related adverse event, including abnormalities in vital signs, was observed [233]. On inspection of the oral cavity only minor mucosal lesions not related to the study medication were seen in seven subjects [233]. Repeated measurements of β-glucan in serum revealed no systemic absorption of the agent following the oral doses of SBG [233]. In saliva, the IgA concentration increased significantly for the highest SBG dose employed [233]. It was concluded that SBG is safe and well tolerated by healthy volunteers, when given orally once daily for 4 consecutive days at doses up to 400 mg [233].

The enhancement of IgA concentration in saliva observed with the highest oral dose of soluble β-glucan in this study is obviously of great interest because of the important contribution it could possibly make to a therapeutic strategy where the main objectives are to try to keep the patient alive until the adaptive immune system can come to the rescue – and where it is important that the rescue forces arrive as soon as possible before it is too late.

There is more than one reason why IgA is important in infectious diseases affecting mucous membranes. IgA not only functions as an extracellular neutralizing antibody (binding viruses), it can do the same even inside infected epithelial cells and may thus help to inhibit viral replication even after the virus has entered an epithelial target cell [234–238].

Extracellular IgA can possibly do more than simply bind and thereby inactivate the virus, so that it cannot bind to the receptors on the plasma membranes of potential targets any more. There are unconfirmed observations suggesting that it can collaborate in a fairly non-specific way with the microbicidal enzyme lactoperoxidase, so that the virus will not only become immobilized, but can also be killed. It has been reported that the antimicrobial effect of the lactoperoxidase system (enzyme with thiocyanate ion and hydrogen peroxide) on Streptococcus mutans was significantly enhanced when the system was combined with secretory IgA [239]. Similar enhancement was observed with lactoperoxidase and myeloma IgA₁ or IgA₂ combinations [239]. This enhancement of the antimicrobial efficiency was not dependent on the presence of specific antibodies to S. mutans in the IgA preparation, but seemed to require binding between lactoperoxidase and immunoglobulin [239]. However, human polyclonal, myeloma IgG or IgM, and rabbit IgG failed to enhance the antibacterial activity of the lactoperoxidase system [239]. None of the immunoglobulins, when added alone, produced antimicrobial effects [239]. Lactoperoxidase was shown to bind to colostral secretory IgA, myeloma IgA₁, IgA₂, and to a lesser degree to monoclonal and polyclonal IgG and monoclonal IgM [239]. This binding was found to have a stabilizing effect on the enzyme activity [239].

It is also known from other studies that microbicidal peroxidases can not only kill bacteria, but also may exert a viricidal effect at least against HIV virus [240–244]. It is reasonable to suppose that the effect may be similar also against other similar viruses, influenza virus included (even if it was not possible to find any experimental study that specifically addresses this question). It should be recognized, however, that chloride (unlike the situation with myeloperoxidase) does not function as an electron donor for lactoperoxidase; instead this enzyme requires bromide, iodide or thiocyanate as a reducing cofactor for microbicidal activity [245]. The viricidal activity of lactoperoxidase is presumably important not only when viruses are bound specifically to corresponding IgA molecules, but also when they are bound to other molecules in the extracellular environment, as for example, when influenza viruses are bound to oligosaccharide groups containing sialic acid in mucin molecules [14]. It may thus be possible that the binding of influenza viruses to mucins may not only represent some form of temporary, but reversible inactivation (because the virus binds to a mucin molecule instead of to a cellular receptor), but also could lead to gradual killing of the virus as a result of the activity of lactoperoxidase and perhaps other microbicidal peroxidases (in combination with H₂O₂ and some appropriate halide or pseudohalide cofactor).

IgA has also been reported to function as an important opsonizing factor in combination with complement, as has been shown with infections involving Pneumocystis carinii [246]. Fc receptors for IgA alone or for IgA + IgM are present on different types of leukocytes, including neutrophils, eosinophils, macrophages, and B lymphocytes [247–251].

Mucosal vaccines aimed at enhancing IgA immunity against influenza have been tested, with some such vaccines using attenuated live influenza virus [252], [253], while other candidate vaccines use non-living influenza virus [254–256]. Such vaccine types (which possibly might be improved even more using better adjuvants) will most likely represent a most significant advance compared with classical intradermal influenza vaccines. Hopefully it may also become possible to produce some of them cheaply enough to make them suitable for mass vaccination even in very poor countries.

The experiment where macaques were infected with Spanish flu virus [35] has shown very clearly that the pathogenesis at least of this form of highly virulent influenza is characterized by a somewhat paradoxical combination of immunodeficiency and immunological over-reaction. Too many of the smart bombs that specifically target the pathogen have been destroyed, and the snipers have all been taken out of action, but large numbers of dumb cluster bombs are still available – for a war theater where most of the enemy soldiers are to be found amidst the civilian population of the country being invaded. So what we are dealing with is a serious weakening of immune reactions dealing with the enemy in combination with a more non-specific form of inflammatory over-reaction that is poorly capable of resisting the enemy, but highly efficient as a cause of harm to its own tissues. However, it may be possible that β-glucans could have therapeutic effects that might help to deal with both of these problems simultaneously, as they might possibly help to weaken the unwanted inflammatory over-reaction at the same time as they may help to improve more effective forms of antiviral response.

Use of taurine as an anti-inflammatory agent?

We shall now try to consider, but much more briefly, what possibly might be done (in addition to what has already been mentioned in our earlier survey article [6]) to enhance the chances of survival for the patient until it is possible for the rescue forces to arrive (i.e. the time it takes to mount a strong enough adaptive immunity response to a new pathogen).

What we need here are substances that can be used as anti-inflammatory agents without causing too much harm to the development of a strong and efficacious anti-pathogen response which we want to come as soon as possible. Some substances that may be protective for the host tissues at the same time as they help to improve antiviral immunity have already been mentioned. Another possible example will be discussed below:

The aminosulfonic acid taurine has multiple physiological roles [6], [257]. It is found at much higher normal concentrations (commonly two orders of magnitude more) in the cytosol of nucleated cells than in blood plasma [257]. Taurine is therefore a suitable candidate for use as a sentinel of plasma membrane damage or disturbance of plasma membrane functions arising, for example, as a consequence of too much oxidative or nitrosative stress, since only a small proportion of the intracellular taurine content leaking out of the cell will be enough to cause a large relative rise in its concentration in the extracellular fluid.

This sentinel function of extracellular taurine can be carried out in two different ways. First, extracellular taurine can bind to receptor sites situated at the outside of the plasma membrane; these receptors may be either GABA receptors (which can use either GABA or taurine as ligands) or glycine receptors (which can use either glycine or taurine as ligands). Or it can react with reactive halogen species (e.g. hypochlorite) formed by microbicidal or parasiticidal peroxidases such as myeloperoxidase, eosinophil peroxidase, and lactoperoxidase that can catalyze the reaction between halide ions (or thiocyanate) and H₂O₂.

Glycine receptors sensitive to taurine have been found on Kupffer cells, i.e. macrophages in the liver [258], where extracellular taurine has an inhibitory action blunting the enhancement of intracellular calcium concentration and TNF-α secretion following stimulation of the cells with LPS [258]. This effect seems to be mediated by a glycine-gated chloride channel [258], so that glycine or taurine binding to the receptor will lead to hyperpolarization of the plasma membrane. It has also been reported that production of TNF-α and superoxide anion radical (respiratory burst) in alveolar macrophages is blunted by glycine [259]; since taurine is an agonist of the glycine receptor in the macrophages [258], it would be expected also to be capable of reducing ROS production by macrophages. Dietary glycine is protective in rat models against endotoxemia, liver ischemia-reperfusion, and liver transplantation, and it is believed that this may be mainly explained by glycine inactivating the Kupffer cell via this newly identified glycine-gated chloride channel [260]. Similarly, it has been reported that taurine (and also betaine) protects rats from LPS hepatotoxicity as measured by changes in aspartate aminotransferase and alanine aminotransferase activities, and total bilirubin levels in serum, and hepatic glutathione contents [261]. LPS challenge increased serum TNF-α and nitrate/nitrite in rats, which were reduced by betaine or taurine intake [261]. Glycine-gated chloride channels have, moreover, also been found in the plasma membrane of neutrophils, where glycine similarly has been shown to blunt the respiratory burst [262]. Thus taurine should be expected to do the same. With neutrophils and monocytes/macrophages both being major players in the pathogenesis of pneumonia induced by hypervirulent influenza, there is good reason to expect that taurine (but also glycine and betaine) might have a similar protective effect in the human lung, as has been demonstrated in the livers of experimental animals.

GABA_B receptors have been found on peripheral nerve fibers, both on C-fibers and at the end of cholinergic visceral neurons [263–266]. GABA_B agonists have inhibitory effects on these nerves, e.g. inhibition of release of substance P from capsaicin-sensitive neurones in the rat trachea [263] and of acetylcholine secretion from cholinergic nerves in the lung and in the colon [264–266], and GABA itself has been shown to inhibit the anaphylactic response in guinea pig trachea [267]. It has been found that there are different subtypes of GABA_B receptors in the central nervous system which differ in their sensitivity to taurine as an agonist (while all of them, of course, are sensitive to GABA). Since the sensitivity of the peripheral GABA_B receptors in relation to taurine (whether it functions as a good agonist or not) has apparently not been studied, one cannot know for certain whether or not taurine may function as a good inhibitor (acting via the GABA_B receptors) in relation to these nerves. But it is known from animal experiments that taurine and homotaurine have antinociceptive properties in experimental pain models where pain is elicited either by heating (hot plate, tail immersion, and tail flick models) or by low pH (injection of acetic acid into the peritoneum) [268–272]. Taurine has also been reported to have beneficial effects in an experimental rat model of asthma, where it significantly reduced the number of eosinophils, reduced lipid hydroperoxide concentration, and reduced Evans blue dye extravasation in bronchoalveolar lavage fluid [273]. A therapeutic effect of taurine (when given as an aerosol spray preparation for inhalation) has been reported for human asthma patients as well; it was of comparable magnitude as for commonly used anti-asthmatic drugs [274]. All of this is also highly compatible with the experience of one of the present authors (O.A.C.), who for several years has been taking high doses of fish powder (about 50 g), which is rich in taurine, as a drug for self-medication of acute hay fever and asthma. The effect (e.g. reduction of nasal secretion) comes very rapidly, in less than 5–10 min, but starts to recede after about 4 h (which is compatible with what is known about the pharmacokinetics of this substance with rapid urinary excretion when the renal threshold is exceeded).

These observations, when taken in combination, are highly suggestive of an inhibitory effect of taurine on C-fibers, which may in turn lead to reduction of the secretion of proinflammatory peptides such as substance P from unmyelinated peripheral nerves, i.e. reduction of neurogenic inflammation (even though it could well be possible that there might be more than one pharmacological target explaining the beneficial effect of taurine observed in rats and humans with asthma, so this is not necessarily the only mechanism). It is therefore possible that taurine also could be used for reducing C-fiber activity in the lungs and blunting the process of neurogenic inflammation (which possibly might represent an important contributory cause of the alveolar edema) in patients suffering from pneumonia caused by hypervirulent influenza. This would be expected not only to lead to reduced extravasation of blood plasma proteins through pores in the venules, as explained earlier, but also less stimulation of leukocytes (i.e. macrophages) by peptides secreted from the C-fibers, as well as reduction of the centrally mediated reflex leading to enhanced ACTH secretion and hence enhanced glucocorticoid secretion as a result of enhanced C-fiber activity.

Taurine can be halogenated by myeloperoxidase to form taurine chloramine, which functions as a potent anti-inflammatory agent [275–280]. Taurine chloramine has been reported to inhibit the production of IL-6 and IL-8 by fibroblast-like synoviocytes in rheumatoid arthritis [275]. It also inhibits inducible NO synthase and TNF-α gene expression, as well as secretion of the chemokines MCP-1 and MIP-2 in alveolar macrophages [276], [277]. The anti-inflammatory effect of taurine chloramine can in part be explained as a consequence of inactivation of the regulatory enzyme IκB kinase, which must be activated in order that the transcription factor NF-κB is activated; taurine chloramine will therefore inhibit NF-κB activation [281–284]. This happens because of oxidation of a methionyl group on the enzyme [281]. Taurine chloramine, moreover, has also been reported to inhibit the activation of Ras following LPS treatment of macrophages and to inhibit ERK1/2 activation in a dose-dependent manner in both RAW 264.7 cells and murine peritoneal macrophages, whereas it did not exert any effect on p38 MAPK activation [283]. But taurine chloramine also has other protective effects, since HOCl and TauCl may directly neutralize IL-6 and several metalloproteinases in the extracellular environment [280].

Administration of taurine to patients suffering from severe H5N1 pneumonia might be done both perorally, by inhalation of an aerosol spray preparation, and by parenteral administration. For conscious patients, it must certainly be most practical to give the substance by the oral route either alone or in combination with other nutrients or drugs. For unconscious or sleeping patients, parenteral administration should probably be preferred when practically possible. The kinetics of the substance with rapid urinary excretion (when given at a high dosage level) should be kept in mind; for oral administration, it might therefore be useful to have a slow-release formulation, ensuring that there will be a high plasma taurine concentration for a more extended period of time. The dosage for severely ill adult patients should probably not be less than about 1 g per day. Taurine appears to be exceptionally non-toxic, which means that the risk of harmful side effects may be judged to be very small, except possibly for insulin-dependent diabetics. This is because taurine has been reported to bind to the insulin receptor [285] and affect blood sugar regulation, at least in experimental animals [286], [287]. Therefore there is a possibility that is more than purely theoretical that administration of high doses of taurine to an insulin-dependent diabetic unfamiliar with the substance might provoke unexpected and potentially dangerous hypoglycemia.

Use of IL-6 receptor antagonist drugs?

The experiment infecting macaques with Spanish flu virus showed that hypersecretion of IL-6 may play a very central role in the pathogenesis of pneumonia caused by hypervirulent influenza, at least in the macaque model [35]. Since there are drugs available, both in the form of mAbs and ordinary drugs that function as IL-6 receptor antagonists [288–290], it would appear logical to test out the efficacy of such drugs in animal models of hypervirulent influenza. Whether they can be used for mass therapy of large numbers of seriously ill patients in the context of poor countries will depend, however, not only on the therapeutic efficacy of these drugs in hypervirulent influenza (which most likely has never been studied), but also on various practical and economic considerations, not least price and how rapidly they can be produced in the amounts that might be required in a pandemic situation. However, it may be possible that such drugs might prove clinically useful for treatment of a manageable number of H5N1 patients in hospital settings during a non-pandemic situation.

Improving organ tolerance against hypoxia and ischemia/reperfusion

Given the severe undersaturation with oxygen in arterial blood that was observed in macaques that had been experimentally infected with the Spanish flu virus [35], it appears logical to attempt not only to treat (or prevent) the alveolar edema (using such methods of inhibiting the proinflammatory activity both of leukocytes and unmeylinated nerve fibers as have already been mentioned), but also to do whatever might be possible to protect various organs against the harmful consequences of inadequate oxygen supply. There is much literature about the protective effect of substances such as taurine [291], [293], selenium [294–297], glutathione [298], [299], melatonin [300–302], and also various other biological antioxidants against tissue damage caused by ischemia/reperfusion in organs such as the heart, kidney, liver, and brain, and in the case of selenium also in global anoxia, with substantial enhancement of the time lag before respiratory arrest and cardiac arrest in response to improved selenium status [303]. Space limitations do not permit an extensive review of this literature here, but much of it is easy to find in medical literature databases such as Pubmed.

Outline of an improved global emergency preparedness strategy

It has already been mentioned how an exceptional mobilization of resources was needed during the Second World War to make penicillin available in such quantities as were needed by American and allied soldiers in the battlefields of the Pacific and Europe [132]. Today, however, we are facing an enemy so strong and so cruel that all the horrors of World War 2 by comparison may look like play in a kindergarten. It is estimated that 55 million people were killed during World War 2, including deaths in Asia [7], while a pandemic of hypervirulent H5N1 potentially might kill 100 times that number or more, if nothing is done to try to stop this enemy before it is too late. And we can be certain that it will already be too late, on the day when the pandemic starts, unless we are well enough prepared in advance.

We have to prepare for a situation that for all practical purposes can only be compared with a full-scale global war situation – if we don't want simply to sit passively and wait for the executioner until we see our own number in the queue. But if we try to think like an experienced general to whom has been entrusted the task of making a good defense strategy, the first thought that must be obvious, at least for a professional general, is that we will need more than one line of defense.

The first defense line should be vaccination combined with highly vigorous efforts to delay the dispersal of the superpathogen as much as possible. Today, however, we are woefully poorly equipped, if the value of that defense is to be anything more than that of the Maginot Line during World War 2. We don't have the quantity of weapons that we need, the weapons are (at least when considering the traditional type of influenza vaccine) far from the quality that we need, we cannot be certain that the enemy will not make himself invisible when he attacks (i.e. we cannot be certain that there will not be another case of successful antigenic drift before the enemy attacks), and if somebody tries to make better weapons, they will often be tempted to make them so sophisticated and also so expensive that it will be economically impossible to produce them in the quantities that are needed.

What is needed here is therefore some kind of coordinated international effort akin to that of the American pharmaceutical industry when it developed penicillin production to the needed industrial scale during World War 2 [132] – but this time we shall need to repeat a similar kind of effort on a global scale. We might also learn something by considering what the human brain is doing, compared with a modern electronic computer. The processes in the brain take place very slowly, compared with the movement of electrons in the computer. But the slowness of serial processes (one step followed by the next step, and so on) is compensated for by an extraordinary capacity for parallel information handling. Which means that our brain is not so poor after all, even when compared with excellent computers, in spite of the extreme slowness of its step-by-step operations judged by the standard of the electronic computer.

So what we need when trying to develop vaccines that are both much better and also cheap enough, is to encourage as much parallel information handling as possible in the form of a large number of parallel research projects. We need many, many different companies and research institutes, each of them with their own vaccine project, while also encouraging diversification so that, collectively speaking, one may try as many different principles and technical solutions as possible. We can then hope that at least one of the participants in this effort of friendly international competition will come up with something that is both so good and so cheap that it can be produced in sufficient quantity to cover the needs of the whole world population.

However, we cannot be confident that the first line of defense will hold. If this should not be the case, it is extremely difficult to see any possible strategy (if we do not simply resign and sit passively while waiting for our own death) except the strategy of counterpandemic with a non-virulent ‘vaccine virus’ also combined with highly vigorous efforts to delay the dispersal of the superpathogen as much as possible [7]. The combination of quarantine/isolation measures and immunization using a live, but low virulence virus should therefore constitute our second line of defense.

The principle is simple enough and easy to understand. However, it will require much careful planning and much practical preparation in advance, like preparing for an ordinary war, if it is to work as we would like it to do (e.g. building up enough stores of such foods as can be used for a quarantine situation over a period of at least 3 months).

The third defense line is therapy for those we are not able to immunize before they are attacked by the deadly virus. But again we need much better weapons, and in much greater quantity, than are available today. Currently recommended therapy can hardly be considered adequate when it cannot prevent the worldwide average lethality from increasing from 43% in 2005 to 69% in 2006, and as much as 82% in Indonesia in 2006.

This paper has hopefully provided documentation good enough for at least drawing the conclusion that there should be no reason for resignation, as far as treatment is concerned. There are so many possible methods of treatment and many substances that appear highly promising on the background of much good research that has already been done, that it would be strange if at least some elements of this might not be used as part of a multifactorial therapeutic intervention strategy that hopefully will work much better than what we have today.

But to test it and document the efficacy of new treatment methods as well as one ideally might desire before translating the scientific information into practical treatment guidelines for possibly billions of patients is again something that hardly can be done without some kind of coordinated international research effort, similar to the program during the Second World War for turning penicillin into a commercially available drug that could be produced in the quantities that were needed.

Abbreviations

Table

ACTH	adrenocorticotrophic hormone
Aδ-fibers	type designation of a group of thin myelinated sensory nerve fibers
AED	androstenediol (formed by hydroxylation of DHEA)
AED-S	androstenediol-sulfate
AET	androstenetriol (formed by hydroxylation of AED)
αMβ2-integrin	see CR3, below
AP-1	activating protein-1 (name of transcription factor)
B220	pan B; name of surface molecule on cells that are precursors of myeloid leukocytes
βGR	β-glucan receptor; same as Dectin-1 receptor
βGR-A	isoform of β-glucan receptor
βGR-B	isoform of β-glucan receptor
B lymphocyte	bursa-derived lymphocyte (name from the lymphoid organ bursa of Fabricius that is found in birds, but not in mammals); different subgroups; gives rise to plasma cells that produce immunoglobulins
C3b	cleavage product formed by degradation of complement factor 3
CB4	Coxsackievirus B4
CCL2	monocyte chemoattractant protein-1 (name of chemokine)
CCL5	monocyte chemoattractant protein-5 (name of chemokine); also called RANTES
CCL20	name of chemokine
CD4^+ T cells	subgroup(s) of T cells best known for their regulatory functions; strongly depleted in AIDS patients; different subgroups of CD4^+ cells are characterized by different cytokine secretion patterns; some of these are called Th1 CD4^+ cells or T-helper 1 cells because they produce mainly Th1-associated cytokines, while others are called called Th2 CD4^+ cells or T-helper 2 cells because they produce mainly Th2-associated cytokines
CD8^+ T cells	subgroup(s) of T cells with killer and regulatory functions; important especially for defense against viruses, against bacterial or eukaryote pathogens that live inside cells of their host (e.g. tubercle bacilli), and defense against cancer cells
CD11b/Mac-1	name of surface molecule on cells that are precursors of myeloid leukocytes, but also on different types of mature leukocytes; same as complement receptor 3 (CR3); binds factor I-cleaved C3b
C-fibers	type designation of mostly unmyelinated nerve fibers, which can have sensory and/or secretory functions (including various subgroups with different functions and properties)
CGRP	calcitonin gene-related peptide (name of one of the peptides secreted from C-fibers)
CI	confidence interval (in statistical or modeling studies)
COX-2	cyclooxygenase-2 (rate-regulatory protein making prostaglandins which is expressed in leukocytes especially when they are activated)
CR3	complement receptor 3; also known as Mac-1, CD11b/CD18, or αMβ2-integrin; binds factor I-cleaved C3b (iC3b)
DC-Chol	name of type of immunoadjuvant (used for enhancing the immunological response to a vaccine)
DC-SIGN	name of receptor found on dendritic cells (DCs); can participate in the uptake of viruses or bacteria by these cells, but also has important regulatory functions
DDX58	same as RIG-I (see below).
Dectin-1 receptor	name of β-glucan receptor found, i.a. on dendritic cells (DCs); not considered to belong to the group of Toll-like receptors, although it has a similar function
DHEA	dihydroepiandrosterone; name of steroid hormone with anti-glucocorticoid action that is produced mainly in the adrenals; also precursor of other signal substances including AED, AED-S, and AET
DNA	deoxyribonucleic acid (made from deoxyribonucleotide monomers)
dsRNAs	double-stranded ribonucleic acids
ERK1/2	extracellular receptor kinase 1/2; name of protein kinase
GABA	γ-aminobutyric acid; name of amino acid that functions as an important inhibitory transmitter substance in the brain
GABAB receptor	subtype(s) of GABA receptor found both in the central nervous system and on peripheral nerves
GSH	reduced glutathione (γ-glutamyl-cysteinyl-glycine)
HIV	human immunodeficiency virus
H1N1	type A influenza virus with hemagglutinin belonging to subgroup 1 and neuraminidase belonging to subgroup 1; common in humans; a highly pathogenic form was responsible for the Spanish flu pandemic
H5N1	type A influenza virus with hemagglutinin belonging to subgroup 5 and neuraminidase belonging to subgroup 1 (as in H1N1 influenzavirus); mainly in birds where it is now often highly pathogenic
H7N7	type A influenza virus with hemagglutinin belonging to subgroup 7 and neuraminidase belonging to subgroup 7; found in birds (sometimes highly pathogenic), only rarely transmitted to humans
HOCl	hypochlorous acid (chemical formula, not abbreviation)
HSV-1	herpes simplex virus 1
iC3b	molecular fragment formed when factor I cleaves C3b; C3b is formed by cleavage of complement factor 3
IFIH1	same as melanoma differentiation-associated gene, also called MDA5
IgA	immunoglobulin A
IgG	immunoglobulin G
IgM	immunoglobulin M
IFN-α	interferon-α
IFN-β	interferon-β
IL-1α	interleukin-1α
IL-2	interleukin-2
IL-3	interleukin-3
IL-4	interleukin-4
IL-6	interleukin-6
IL-6R	interleukin-6 receptor
IL-8	interleukin-8
IL-10	interleukin-10
IL-11	interleukin-11
IL-12	interleukin-12
IP-10	name of chemokine
IRF-3	name of transcription factor
ITAMs	immunoreceptor tyrosine-based activation motifs
Jak1	Janus kinase 1 (name of tyrosine kinase)
Jak2	Janus kinase 2 (name of tyrosine kinase)
LD50	lethal dose 50; dose of toxic substance that is sufficient to kill half of the animals used for testing its toxicity
LD75	lethal dose 75; used for dose of virus that causes 75% of the animals used in the experiment to die
LD100	lethal dose 100; used for dose of virus that causes 100% of the animals used in the experiment to die
LPS	lipopolysaccharide; name of group of molecules found in bacterial cell walls; recognized by Toll-like receptors; also called endotoxin
MCP-1	name of chemokine
MIP-2	name of chemokine
mRNA	messenger RNA (ribonucleic acid)
MDA5	same as IFH1
MIO	melatonin-induced opioid; type of opioid peptide(s) produced by leukocytes when they are stimulated by melatonin
n	number of cases included in a statistical study
NFAT	nuclear factor of activated T cells (name of transcription factor)
NF-κB	nuclear factor κB (name of transcription factor).
N-glycosylation	glycosylation at nitrogen atom
NK cells	natural killer cells; name of cell type considered part of the innate immune system with important effector as well as regulatory functions; participate in the killing of virally infected cells, of cells infected by bacteria and of cancer cells; participate also (by means of cytokine secretion) in the control of class switching by B lymphocytes
NO	nitric oxide (used for regulatory purposes and also as a weapon for killing pathogenic invaders or cancer cells)
NS1	non-specific 1 (name of one of the internal proteins of influenza virus)
OMp	oxidative macrophages, i.e. macrophages that contain little GSH
p38 MAPK	p38 mitogen-activated protein kinase; one of the MAPKs
PGE2	prostaglandin E2 (made from arachidonic acid)
PGF2α	prostaglandin F2α (made from arachidonic acid)
PGI2	prostaglandin I2 (made from arachidonic acid); also called prostacyclin
PIAS	protein inhibitor of activated STATs
3pRNA	uncapped 5′-triphosphate RNA
R	reproductive number (average number of other persons that will be infected by one infected person)
RANTES	name of chemokine; same as CCL5
RIG-I	retinoid acid-inducible protein-I; also called DDX58
RNA	ribonucleic acid (made from ribonucleotide monomers)
ROS	reactive oxygen species (such as H2O2, superoxide anion radical, hydroxyl radical, and singlet oxygen)
RMp	reductive macrophages, i.e. macrophages that contain much GSH
SARS	severe acute respiratory syndrome (name of disease and of the coronavirus that causes this disease)
SBG	soluble branched yeast β-1,3-D-glucan
sgp130	soluble glycoprotein 130
SHP-2	name of tyrosine phosphatase (reversing the effect of tyrosine kinases)
sIL6-R	soluble interleukin-6 receptor
SOCS	suppressor of cytokine signaling (name of inhibitor of Janus kinase)
Src	name of tyrosine kinase
ssRNA	single-stranded ribonucleic acid
STAT-1	signal transducer and activator of transcription-1
STAT-2	signal transducer and activator of transcription-2
Syk	name of tyrosine kinase
syk-/- cells	gene-manipulated cells that are doubly deficient in the gene for Syk
TauCl	taurine chloramine
T cell	thymus-derived lymphocyte
TGF-β	transforming growth factor-β
Th1	T-helper 1 (for particular subgroups of T cells and cytokines)
Th2	T-helper 2 (for particular subgroups of T cells and cytokines)
TLR-2	Toll-like receptor-2
TNF-α	tumor necrosis factor-α
Toll-like receptors	group of receptors used for recognition of molecules found in large groups of pathogenic organisms or viruses, but not normally in human tissues or cells (e.g. structural molecules found in bacterial cell walls, bacterial and viral DNA); found also in plants, and must therefore be phylogenetically very ancient (earlier than the departure of animal and plant ancestors to form two separate evolutionary lineages perhaps more than 2 billion years ago); echinoderms have been found to contain one order of magnitude more different types of Toll-like receptors than human cells

Acknowledgements

We wish to thank Professor Nevin S. Scrimshaw and Professor John G. Ormerod for their criticism and helpful suggestions during the preparation of the manuscript (without making them responsible either for all of the opinions that are expressed here or for those factual errors that possibly might have crept in despite efforts to try to avoid them). We also thank John F. Moxnes for several useful discussions before this manuscript was written.