Modern diets and diseases: NO–zinc balance

Medical Hypotheses (1999) 53(1), 6–16 © 1999 Harcourt Publishers Ltd Article No. mehy.1999.0867

Modern diets and diseases: NO–zinc balance Under Th1, zinc and nitrogen monoxide (NO) collectively protect against viruses, AIDS, autoimmunity, diabetes, allergies, asthma, infectious diseases, atherosclerosis and cancer J. E. Sprietsma Bennekom, The Netherlands

Summary Thanks to progress in zinc research, it is now possible to describe in more detail how zinc ions (Zn++) and nitrogen monoxide (NO), together with glutathione (GSH) and its oxidized form, GSSG, help to regulate immune responses to antigens. NO appears to be able to liberate Zn++ from metallothionein (MT), an intracellular storage molecule for metal ions such as zinc (Zn++) and copper (Cu++). Both Zn++ and Cu++ show a concentrationdependent inactivation of a protease essential for the proliferation of the AIDS virus HIV-1, while zinc can help prevent diabetes complications through its intracellular activation of the enzyme sorbitol dehydrogenase (SDH). A Zn++ deficiency can lead to a premature transition from efficient Th1-dependent cellular antiviral immune functions to Th2dependent humoral immune functions. Deficiencies of Zn++, NO and/or GSH shift the Th1/Th2 balance towards Th2, as do deficiencies of any of the essential nutrients (ENs) – a group that includes methionine, cysteine, arginine, vitamins A, B, C and E, zinc and selenium (Se) – because these are necessary for the synthesis and maintenance of sufficient amounts of GSH, MT and NO. Via the Th1/Th2 balance, Zn++, NO, MT and GSH collectively determine the progress and outcome of many diseases. Disregulation of the Th1/Th2 balance is responsible for autoimmune disorders such as diabetes mellitus. Under Th2, levels of interleukin-4 (Il-4), Il-6, Il-10, leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) are raised, while levels of Il-2, Zn++, NO and other substances are lowered. This makes things easier for viruses like HIV-1 which multiply in Th2 cells but rarely, if ever, in Th1 cells. AIDS viruses (HIVs) enter immune cells with the aid of the CD4 cell surface receptor in combination with a number of co-receptors which include CCR3, CCR5 and CXCR4. Remarkably, the cell surface receptor for LTB4 (BLTR) also seems to act as a co-receptor for CD4, which helps HIVs to infect immune cells. The Th2 cytokine Il-4 increases the number of CXCR4 and BLTR co-receptors, as a result of which, under Th2, the HIV strains that infect immune cells are precisely those that are best able to accelerate the AIDS disease process. The Il-4 released under Th2 therefore not only promotes the production of more HIVs and the rate at which they infect immune cells, it also stimulates selection for the more virulent strains. Zn++ inhibit LTB4 production and numbers of LTB4 receptors (BLTRs) in a concentration-dependent way. Zn++ help cells to keep their LTB4 ‘doors’ shut against the more virulent strains of HIV. Moreover, a sufficiency of Zn++ and NO prevents a shift of the Th1/Th2 balance towards Th2 and thereby slows the proliferation of HIV, which it also does by inactivating the HIV protease. Research makes it look likely that deficiencies of ENs such as zinc promote the proliferation of Th2 cells at the expense of Th1 cells. Zinc deficiency also promotes cancer. Under the influence of Th1 cells, zinc inhibits the growth of tumours by activating the endogenous tumour-suppressor endostatin, which inhibits angiogenesis. Received 11 March 1999 Accepted 22 March 1999 Correspondence to: J. E. Sprietsma, Van Rouwenoortstraat 6, 6721 TV Bennekom, The Netherlands. Fax: +31 (0)318 416214.

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The modern Western diet, with its excess of refined products such as sugar, alcohol and fats, often contains, per calorie, a deficiency of ENs such as zinc, selenium and vitamins A, B, C and E, which results in disturbed immune functions, a shifted Th1/Th2 balance, chronic (viral) infections, obesity, atherosclerosis, autoimmunity, allergies and cancer. In view of this, an optimization of dietary composition would seem to give the best chance of beating (viral) epidemics and common (chronic) diseases at a realistic price.

INTRODUCTION Despite the best efforts of many, AIDS still threatens to develop into an epidemic of dramatic proportions, particularly in less developed countries. In sub-Saharan Africa, more than 5000 people die of AIDS every day, and about four million new cases emerge each year. In 1998, onequarter of the adult population of Botswana was HIVinfected. AIDS is also on the rise in Asia and Russia. In the rich Western world, however, the number of fatalities fell by two-thirds between 1995 and 1997 as a result of new medicines such as the very expensive synthetic protease inhibitors, while the number of new infections has stabilized at about 75 000 a year. Unfortunately, use of this therapy elsewhere is not at all a realistic prospect given the cost of drugs and the medical facilities that are needed. Medical and other aid organizations thus find themselves empty-handed where help is most needed. HIV-infected people often seem to have shortages of ENs such as vitamins A, B12, C and β-carotene, and of trace elements, including selenium (Se) and zinc (Zn). This undermines the effectiveness of their immune systems. Supplementing one or more of these ENs appears to be able to improve the immune functions of pregnant women and reduce the risk of transmitting HIV to their children (1). An optimal diet which, in addition to providing an adequate caloric input, also contains sufficient ENs per calorie seems to offer a more hopeful and realistic prospect. Optimal immune functions have, after all, been protecting man and beast against death from pathogenic diseases for millions of years. The close relationship between diet and immune function will be briefly illustrated in this article for a number of ENs that have been the subject of a great deal of scientific research. Zinc was once chosen more or less by accident as primus inter pares for these ENs (2–8).

and with each other for ENs. (2–12). As in many of these proteases, the amino acid cysteine interacts with Zn++ in many cellular control mechanisms, which frequently involve zinc–finger–type protein molecules. Zn++ and cysteine together direct cellular processes via concentrationdependent structural changes. The activity of HIV–1 protease is also regulated by Zn++–induced changes to cysteine (13,14). The activity of the enzyme sorbitol dehydrogenase (SDH) – which is involved in diabetes complications such as retinopathy – is, for example, regulated by Zn++ and glutathione (GSH) (15). In the intracellular environment, Zn++ are mostly bound to the zinc storage molecule metallothionein (MT) (15). The balance between GSH and its oxidized form, GSSG, determines how many Zn++ are transferred by MT to, for instance, SDH (15–18). GSH helps MT to retain Zn++, while GSSG makes more Zn++ available. Sufficient active SDH will remain available provided that there are enough GSH/ GSSG and MT-bound Zn++ and that enough energy is available in the cells in the form of adenosine triphosphate (ATP) (15–19a). ATP also seems to be required for the transfer of Zn++ to other intracellular molecules via combination with MT and GSH/GSSG (19). Zn++ released from MT are available for concentration-dependent activation of enzymes like SDH, while HIV protease is inactivated by both Zn++ and Cu++ (5–10,15–21). Cells can keep SDH active and HIV–1 protease inactive provided that Zn++, Cu++, MT, GSH, GSSG, Se, methionine, cysteine and cystine are sufficiently available, along with vitamins C and E, which are closely involved in the synthesis and functioning of MT, GSH and GSSG (5–10,15–21). However, in the presence of an HIV infection, deficiencies occur of inter alia cysteine and GSH, while extra GSH and cysteine can inhibit the multiplication of HIVs (5–10,22–24). The survival period of HIV infections is shortened by GSH deficiency, while extra cysteine and/or GSH lengthens it (5–10,25–28).

ZINC (ZN), COPPER (CU) AND HIV-1 PROTEASE

NITROGEN MONOXIDE (NO) ORGANIZES RESISTANCE TO ANTIGENS

When retroviruses infect host cells, they direct the synthesis of precursor proteins from which their own – often cysteine – proteases and, later, the components of new virus particles are excised. There are countless types of virus and each has its own more or less complex replication procedures which compete with the host cell

NO regulates a number of cellular and physiological functions, including cellular communication, blood pressure, coagulation, memory and immune functions (29,29a). Apart from the excess found in acute or chronic disease states, NO is almost exclusively involved in defending the body against pathogens (30–33). Macrophages, which are

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often the first cells to signal the presence of antigens from food and the environment and to help eliminate them, depend for optimal functioning on NO generated from L-arginine by their NO-synthase (NOS). If disease or malnutrition leads to a shortage of arginine, the result can be an excess of NO/NOS-derived reactive oxygen intermediates (ROIs). Thse can cause damage to neighbouring cells and tissues (34,34a,34b). Extensive biochemical research into the com-plex nitrosylation activities of NO is currently underway (29–37). NO AND DIABETES MELLITUS Previously, it was assumed that damage to β-cells in the pancreas was primarily attributable to NO, but it now increasingly appears that a shortage of ENs such as zinc, methionine and arginine also plays a major role. Shortages of arginine can certainly produce local surpluses of ROIs – for example as the result of (chronic) (viral) infections (34,34a,34b). Various forms of stress, such as antigenic infections, cause immune cells to bring into circulation interleukin-1 (Il-1) and interleukin-6 (Il-6), causing a lot of zinc to migrate to the liver and kidneys (as this is where extra MT is then synthesized) (5,6). Il-1 and Il-6 cause the liver to ‘board’ zinc (2–6). In an already zinc-depleted body, Il-1 and Il-6 increase the storage of zinc even more by increasing the rate of synthesis of MT in the liver, kidneys and intestines (38,39). As a result, disease and other sources of stress cause an already zinc-deficient body to keep perhaps even more zinc away from the insulin-producing β-cells precisely when they need extra zinc. This has long been suspected and has now been more or less demonstrated (2–4,40). Normally, ZnMT complexes, together with other antioxidants, offer the cells adequate protection against ROIs, metabolites produced via, inter alia, NO/NOS (2,4–8,10, 29–35). When, in chronic disease states which might result from (viral) infections or from EN deficiencies, an excess of ROIs occurs around the β-cells, diabetes could be the final result of the damage done to these β-cells by the ROIs (34). The β-cells can suffer damage because there are insufficient antioxidants in the β-cells to counter the increased ROI levels (2–5). A sufficiency of zinc in β-cells, however, reduces insulin secretion and thereby the amount of zinc leaving the β-cells with the insulin. However, the longer overproduction of insulin continues, the fewer antioxidant ZnMT complexes remain in the β-cells. This is partly the result of the alreadyexisting shortage of insulin release-inhibiting zinc in the β-cells (2–10,40,42,44). Persistent (viral) infections may lead to diabetes by way of deficiences of Zn++, MT, GSH, methionine, cysteine, arginine, Se, vitamins C and E, and an excess of ROIs. An excess of ROIs can be generated enzymatically from Medical Hypotheses (1999) 53(1), 6–16

NOS and L-arginine and non-enzymatically from direct reactions between H2O2 and L-arginine (34,34a,34b,34c). NO INACTIVATES HIV-1 PROTEASE NO inactivates HIV-1 protease, just as it helps activate or inactivate all other kinds of cysteine enzymes (29,36, 36a,37,45,46). These effects of NO result from, inter alia, the transfer of Zn++ to or from these enzymes. Within cells, the GSH/GSSG and Zn++MT/T equilibria also play an important role in this. Thioneine (T) is MT from which most Zn++ have been removed (6,15–19). The intracellular balance between GSH and GSSG depends on the antioxidant potential in these cells which, in turn, is determined by the availability of, among other things, an adequate supply of vitamin C and E, enzymes such as catalase, ZnCuSOD, MnSOD, and ZnMT complexes (2–10,41–43,47–50). NO CAN RELEASE ZN++ IN CELLS NO can release Zn++ from ZnMT complexes in the cytoplasm and nuclei of cells (51,52). Immune cells such as neutrophils can also release oxidants that can free Zn++ from protein molecules such as MT (53,54). Zn++ inhibit and Cu++ block the activity of HIV-1 protease (5–10). As long as sufficient Zn++ are made available to the protease from MT, this HIV-1 protease remains inactive because it can only become active when enough Zn++ are removed from it (5–10,15–21,55). Both zinc and NO acting separately are able to inactivate HIV-1 protease, but in practice they are probably more effective acting together (5–10,15–29). As long as sufficient Zn++, Cu++, Se, GSH, GSSG, MT, methionine, cysteine, arginine and vitamins C and E (necessary for the synthesis and maintenance of GSH, MT and NO) are available in an HIV-1 infected cell, they can effectively suppress the virus by keeping its protease inactive (5–10,15–37). ZINC DEFICIENCY MOVES THE TH1/TH2 SWITCH TOWARDS TH2 In previous articles, a close relationship was shown to exist between a sufficiency of Zn++ and an optimally functioning immune system. Under the influence of a zinc deficiency, immune functions change from a Th1 cell-based, predominantly cellular response to a Th2based, predominantly humoral, response, as a result of which various diseases follow a less favourable course (2–10). Patients with head and neck cancer are, before their disease manifests itself, often significantly low in zinc and their Th1/Th2 balance is shifted towards Th2 (56). The more zinc-deficient they were, the worse the progress of their disease – their tumours were larger, © 1999 Harcourt Publishers Ltd

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they were more frequently hospitalized for other illnesses and died earlier (56). GSH DEFICIENCY MOVES THE TH1/TH2 SWITCH TOWARDS TH2 The GSH level in antigen-presenting cells (APCs) – immune cells that present antigens to T-helper cells (Th) – determines whether these helper cells will develop into Th1 or Th2 cells (26). The greater the GSH deficiency in the APCs, the greater the shift in the helper cell balance towards Th2 cells (26). Many human diseases are associated with zinc and GSH deficiencies (2–10,26,56). Cancers and AIDS in particular show a significantly less favourable development in the face of these deficiencies (2–10,22–28,56). In contrast with what was believed until recently, it appears to be not so much the type of antigen as the presence or absence of a GSH deficiency in APCs that determines whether T helper cells will develop into Th1 or Th2 cells to combat those antigens (56). Other factors that determine the Th1/Th2 switch almost certainly include the numbers in which, the period over which, and the manner by which APCs present the antigens to the T cells (5,6). After all, the supply and reserves of ENs during exposure to an antigen will determine to a large degree whether, after some time, a temporary and/or local deficiency could arise of, for example, GSH, NO, and/or Zn++, which could bias the Th1/Th2 switch towards Th2 cells (5–10,26,56). This is clearly an important issue when it is realized that malaria alone affects 250–400 million people each year, resulting in a million deaths; and that in malaria, just as with AIDS, tuberculosis, leprosy and allergic disorders, the course of the disease is determined to an important extent by the Th1/Th2 switch (5–8,56–58) EN DEFICIENCY MOVES THE TH1/TH2 SWITCH TOWARDS TH2 Immature, naive T-cells, i.e. cells that are not yet directed towards a particular antigen, will, if cultured over successive days in the same medium, initially produce very little Il-4, but then progressively more with each cell division. The production of Il-2, on the other hand, proceeds in the opposite direction by starting high and then declining. Although part of a complex whole, one thing is clear – as more cells divide and grow in a culture medium, the availability of ENs for all cells in that medium diminishes with each successive cell division, and the cells will have fewer ENs such as zinc available. The number of cell divisions and/or the falling availability of ENs appears to transform these naive T-helper cells from Th1 to Th2, as determined by the transition from predominantly Il-2 to predominantly Il-4 (58,59). EN © 1999 Harcourt Publishers Ltd

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deficiencies thus appear to push the Th1/Th2 balance towards Th2. However, it is not clear to what extent the number of cell divisions also plays a role in this. NO AND ZINC CONTROL THE TH1/TH2 BALANCE As soon as Th1 cells are activated by ‘their own’ antigen, they release larger quantities of NO. On the other hand, Th2 cells activated by the same antigen do not release any NO at all (60). The NO released by Th1 cells then inhibits the release of Il-2 and γ-interferon by these same Th1 cells. For Th1 cells, therefore, NO forms a link in a feedback mechanism which ensures that multiplication and activation of the Th1 cells ceases after a certain period (2–10,60). NO has no influence on the production of Il-4 by Th2 cells. Th1 cells exposed to the NO released by their own activity thus cease their activity after a suitable delay. This activity may consist , for instance, of clearing up virus-infected β-cells (2–10,60). Should cytotoxic T-lymphocytes (CTLs) under Th1 remain active for too long against antigens bound to β-cells, autoimmunity and diabetes could result (2–4). Where there is a shortage of arginine, macrophages will produce less NO and more ROIs (34,34a,34b). Zincdeficient β-cells are vulnerable to ROIs and will therefore become damaged earlier and more often (2–5,29–35,38–40). Deficiencies of both zinc and arginine therefore make β-cells more vulnerable (2–6,38–40). A sufficiency of NO timely inactivates CTLs under Th1 and/or strongly reduces their numbers (60). Il-12 is essential for the multiplication of CTLs under Th1 and is released by activated macrophages that fight, for example, virus infections in β-cells. The macrophages will stop releasing Il-12 when, for instance, they no longer find enough viral antigen in the neighbourhood of the β-cells, and the number of active CTLs consequently falls (61). In order to prevent unnecessary damage to healthy β-cells by a surplus of ROIs, macrophages need to produce enough NO and cease releasing Il-12 at the appropriate time in order to allow the number of CTLs to fall (60,61). The release of Il-12 by macrophages is inhibited by PGE2, which raises the level of cyclic AMP (cAMP) in macrophages (60,62). The intracellular cAMP level is regulated by the zinc enzyme phosphodiesterase-4A (PDE4A), which, when activated, breaks down cyclic AMP, thus reducing its cellular level (63). PDE4A is activated by very low levels of intracellular free Zn++ and inactivated by higher concentrations (63). Higher concentrations of free Zn++ in macrophages will therefore raise cAMP levels, halting the release of Il-12. Higher levels of free Zn++ therefore inhibit, via increased cAMP levels, the release of Il-12 by macrophages, causing CTLs under Th1 to fall in number and activity in places where Medical Hypotheses (1999) 53(1), 6–16

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they are fighting (viral) infections. NO releases Zn++ from MT as soon as it is available in sufficient quantities within the cells (51–54). Enough NO will down-regulate the release of Il-12 from macrophages via raised free Zn++ and cAMP levels (61–63). In this way, zinc and NO together control the Th1/Th2 balance (61–63). In addition to this, NO is able to protect endothelial cells against both apoptosis and the cytotoxic action of tumour necrosis factor-α (TNF-α) via an increase in cAMP and/or cGMP (64–66a). It is likely that in this way NO and zinc collectively protect β-cells and hepatocytes against ROIs as well (35,45,46,61–66a). DISRUPTION OF THE TH1/TH2 BALANCE AND AUTOIMMUNITY/DIABETES Zinc deficiency moves the Th1/Th2 balance towards Th2 (5–8,10). Under Th2, immune cells bring more Il-4, Il-6, Il-10 and PGE2 in circulation (2–10). Il-4, Il-10 and PGE2 lower the production of NO by macrophages because they activate the enzyme arginase, which then breaks down the macrophages’ arginine (68). Under Th2, macrophages are inactivated and their NO production falls off as a result of the shortage of intracellular arginine brought about under these conditions (67,68). With the fall in NO production, the concentration of ROIs rises in the vicinity of already weakened pancreatic β-cells where, under a predominance of Th2 activity against an (viral) infection, a more or less chronic inflammation can arise (2–6,10,29–35,38–40). In addition, under a predominance of Th2 cell activity, those β-cells with lower levels of the antioxidant ZnMT complexes and a deficiency of NO – which protect them against apoptosis and cytokines like TNF-α – will more often suffer damage and could die altogether (2–6,29–40,60,64–66a). As a result of this higher rate of cell death through necrosis and apoptosis, the immune system will be exposed to a combination of viral and as yet unfamiliar autoantigens. CTLs specific for these ‘newly discovered’ (auto) antigens will, once under Th1 control, go to work and eliminate the antigens that were as yet foreign to them. In this way, new CTLs are recruited for (auto) immune activities. These CTLs would normally become less active in time as a result of the locally increased NO production by macrophages, if not for the fact that under Th2 the release of NO from macrophages is inhibited by the higher levels of Il-4, Il-10 and PGE2 (60). Zn++ and a sufficient NO level are both involved in the timely inhibition of Il-12 release by these macrophages (51–54,60–63). If, as a result of NO deficiencies and Il-4 surpluses from a local Th2 dominance, macrophages cease their Il-12 release too late for the CTLs that have become involved in these (auto) immune processes and that are operating locally under the Th1 banner, these Medical Hypotheses (1999) 53(1), 6–16

CTLs will continue to be able to keep the (auto)immune process going for some time, especially locally. Allowing for inevitable differences, something like this may underlie autoimmune processes other than those involved in diabetes. Shortages of ENs such as Zn, Se, cysteine and vitamins C and E disrupt the Th1/Th2 balance, as a result of which, albeit locally and only for certain periods, CTLs functioning under Th1 could start to repeatedly combat as yet unfamiliar (auto) antigens released from dying cells. As a result of an excess of Il-4, Il-10 and/or PGE2 and a shortage of arginine and NO, combined with an excess of ROIs, this process continues on average for longer than is desirable or necessary and leads to autoimmune processes. The enzymatically (via NOS) and non-enzymatically generated excess of ROIs will then repeatedly damage the surrounding cells and tissues (34,34a,34b,34c,34d,51–54, 60–68). The characteristic vicious circles of autoimmunity are the result.

LEUKOTRIENES, PROSTAGLANDINS AND IMMUNE REACTIONS UNDER TH1/TH2 Polymorphonuclear leukocytes (PMNLs) such as neutrophilic granulocytes are important producers of leukotrienes such as LTA4 – the direct precursor of the potent pro-inflammatory LTB4 (69). Through their enzymes 5-lipoxygenase (5-LO) and LTA4 hydrolase, activated PMNLs release arachidonic acid from cell membranes and subsequently convert the LTA4 into LTB4 (69–72). These form part of regulation systems about which a lot but certainly not everything is known. During the early phase of an inflammatory reaction, PGE2 is produced in a way comparable to LTB4. Together, LTB4 and PGE2 help to regulate T-cell activity under Th1 and/or Th2 (72). PGE2 mainly serves to inhibit T-helper cells (Th1), and LTB4 inhibits Th1 cells while stimulating T-suppressor cells to greater activity. In this way, PGE2 indirectly inhibits the release of Il-2 and γ-interferon (both primarely made by Th1 cells) while having no effect on the production of Il-4 or Il-5 by Th2 cells (71).

LTB4 AND PGE2 MOVE THE TH1/TH2 SWITCH TOWARDS TH2 LTB4 and PGE2 increase the levels of Il-4 and Il-5, which in turn promote the production of IgE (71). LTB4 and PGE2 are closely involved in the generation and maintenance of allergies under Th2 and in the inhibition of Th1 immune reactions (58–62,71). Raised LTB4 and PGE2 levels are found in asthma, arthritis, psoriasis, ulcerative colitis, inflammatory bowel disease (IBD), Crohn’s disease, (food) allergies, cancer and AIDS (2–10,56,61–63,73–76). © 1999 Harcourt Publishers Ltd

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HIV-1 MULTIPLIES IN TH2 BUT NOT IN TH1 CELLS In the course of the disease process that leads to AIDS, immune functions move progressively from Th1 to Th2. There is a mouse model for AIDS called murine AIDS (MAIDS) which is caused by the murine leukaemia virus (MLV) (77). MAIDS develops in T-cells that carry the CD4 receptor in their cell membranes. MAIDS only takes hold and kills when the host’s immune functions move sufficiently into the Th2 phase, which is characterized by raised levels of Il-4. Mice that are unable to produce adequate amounts of Il-4 do not go on to develop MAIDS (77). Something similar also happens in humans. The further the AIDS process develops, the more the immune response moves into the Th2 phase (5–10,77–79). Research has shown that HIVs multiply almost exclusively in Th2 cells and rarely, if ever, in Th1 cells (5,6,78,79). In Th2 cells, apoptosis occurs to such an extent that the number of Th1 and Th2 cells remains about equal (5,78,79). Recent research has corroborated that HIV viruses multiply predominantly in memory T-cells and almost never in naive T-cells (CD45RA+/CD4+) (80). It is mainly T-cells and monocytes/macrophages with CD4 in their cell membranes that become infected with HIVs and die off as the disease progresses (80–83). Together with CD4, co-receptors such as CCR3, CCR5 and CXCR4 also play a major role in cellular infection by HIVs (5–10,58,77–86).

THE TH2 CYTOKINE IL-4 PLAYS A CRITICAL ROLE IN HIV SELECTION Il-4 shifts the immune reponse phase from Th1 to Th2, with the result that, unlike under Th1, viruses like HIVs can no longer be countered directly and effectively by CTLs. Moreover, HIV viruses are more prone to multiply in Th2 than in Th1 cells (5,6,77–84). Under Th2, therefore, more HIV viruses will be produced, while fewer are cleared up. In view of this, researchers have sought ways to favour Th1 at the expense of Th2. Repeated subcutaneous injections of the Th1 cytokine Il-2 have led to more or less promising results (87). As a result of this extra Il-2, immature, naive T-cells CD45RA/CD4+ multiplied in AIDS patients and were able to produce more Il-2 as well as Il-4 (87). Herein, however, may lie a drawback of this Il-2 approach, namely that the production of extra Il-4 is definitely undesirable. Recent research has again underlined how the release of Il-4 under Th2 conditions leads to the selection of specifically those HIV strains that most strongly promote the disease process in AIDS (88,89,89a). Il-4 promotes the production of the above-mentioned CXCR4 co-receptor that allows the more virulent strains of HIV to infect cells while © 1999 Harcourt Publishers Ltd

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inhibiting the expression of the CCR5 receptor in CD4+ T—cells (58,88). HIV viruses that mainly use CXCR4 and CD4 as a ‘cell door’ now appear to accelerate the development of the AIDS disease process (88,89). Moreover, the protein gp120, which is released by HIV-1, binds to the T-cell receptors CD4 and CXCR4, causing CD4+ T-cells to die through apoptosis (58a). The switch from Th1 to Th2 via Il-4 thus leads, in general, to a faster proliferation of HIVs and selection of more virulent HIV types, thus accelerating the development of AIDS. In addition, cell death as a result of apoptosis triggered by the binding of gp120 to CD4/ CXCR4 receptors will also increase (58,58a,88,89,89a).

THE LTB4 RECEPTOR (BLTR) ALSO PROVIDES HIV-1 WITH ACCESS TO CELLS In addition to the co-receptors CCR3, CCR5 and CXCR4, CD4+immune cells have yet another co-receptor that allows HIV to infect cells (90). This newly discovered co-receptor, BLTR, normally functions as a receptor for LTB4, a leukotriene that is involved in promoting inflammatory reactions in general, and that will, as such, be present in almost all infections. BLTR is, like CXCR4, a CD4 co-receptor that preferentially admits the more aggressive HIV-1 viruses to the cells (88–90a). This functioning of BLTR as a co-receptor for HIV-1 was not confirmed in a recent investigation using another set of HIV strains and experimental procedures (90a). Additional work is needed to clarify some differences (90a). BLTR and CXCR4 both help to accelerate the AIDS disease process (58,58a,88–91). It has been shown above that, for various diseases, LTB4 and PGE2 together bring more Il-4 and Il-5 into circulation and shift the Th1/Th2 balance in favour of Th2, as a result of which these diseases take on a more chronic character and develop less favourably (2,5,6,10, 58–62,71–77). Remarkably, it seems that BLTR, the receptor for LTB4 – a substance released in countless infectious diseases, in particular under Th2 – is used by HIV to accelerate the disease process (88–91).

ZINC INHIBITS LTB4 PRODUCTION IN A CONCENTRATION-DEPENDENT MANNER The production of LTB4 involves the enzymes 5-lipoxygenase (5-LO) and LTA4-hydrolase (69–72). Both of these are zinc enzymes (92). LTA4-hydrolase needs a certain amount of zinc to convert LTA4 into LTB4 but is inhibited if the zinc concentration rises above 10 µM. If the enzyme-inhibiting quantity of Zn++ is removed from LTA4-hydrolase with Zn++-binding substances such as EDTA or dipicolinic acid, the enzyme regains its activity. Medical Hypotheses (1999) 53(1), 6–16

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Zn++ inactivation of LTA4-hydrolase is therefore reversible (92). Low concentrations of free Zn++ activate, and higher concentrations inhibit the activity of LTA4hydrolase reversibly. The activity of the enzyme 5-LO, which releases from cell membranes the arachidonic acid needed to make LTA4 and, subsequently, LTB4, is also zinc-dependent but is reversibly inhibited at lower concentrations of free Zn++ (> or =5 µM). Cu++ also inhibit 5-LO activity but in this case irreversibly (93). In other words, higher concentrations of free Zn++ inhibit the activity of enzymes that release more LTB4. Lower concentrations of free Zn++ raise and higher concentrations inhibit the production of LTB4 and do so within narrow limits (92,93). Zinc helps to downregulate the inflammatory symptoms that accompany diseases such as atherosclerosis, partly by inhibiting 5-LO (2,3,5,6,10,58–62,69–77,88–93). It has already been shown how zinc, MT, GSH and NO collaborate closely to regulate the availability of Zn++ and their transfer within cells (5–10,15–21,51–55). Shortages of zinc, MT. NO and/or GSH and/or the ENs from which they are made will sooner or later lead to the disregulation of these finely balanced control systems. Zn++ activate or inhibit in a concentration-dependent way, and within narrow limits, the production of LTB4. Zinc is therefore a trace element involved to an important degree in whether or not the Th1/Th2 balance is shifted in the direction of Th2 as a result of a shortage of zinc and the resulting increase in Il-4 levels (5–10,22–28,56, 58,59,71,77–79,90–93). Should a situation arise in cells in which NO is no longer able to release sufficient Zn++ from ZnMT complexes to inhibit 5-LO activity in time, this could result in an increase in LTB4 production, as under Th2. Deficiencies of one or more of the above ENs can therefore lead to the raised LTB4 levels that play a major role in all kinds of chronic (infectious) diseases and allergies (50–76,92,92a,93). ZINC REDUCES THE NUMBER OF LTB4 RECEPTORS (BLTR) BLTR receptors occur on the surface of leukocytes such as neutrophils, eosinophils, monocytes and macrophages and are involved in the regulation of various inflammatory reactions. Research has shown that the metal ions magnesium (Mg++), calcium (Ca++), manganese (Mn++) and cobalt (C0++) all increase the number of BLTRs in the cell membranes of these leukocytes. Zn++, on the other hand, are able to reduce the number of BLTRs without affecting the affinity of LTB4 for the remaining BLTRs. Unlike Mg++, Ca++, Mn++ and Co++, only Zn++ seem able to lower LTB4’s capacity to bind to leukocytes (94). Zinc is able to reduce the number of BLTR receptors Medical Hypotheses (1999) 53(1), 6–16

by a factor of three. Having an adequate supply of zinc in and around leukocytes during an HIV infection therefore seems to be important in keeping the LTB4 ‘doors’ closed against HIV-1 viruses. ZN++ AND NO PROTECT AGAINST INFECTIOUS DISEASES, AUTOIMMUNITY, ALLERGIES AND CANCER UNDER TH1 A rapidly growing body of research has shown that immune activities under Th1 can, with the aid of Zn++ and NO, offer protection against a broad range of clinical pictures that include (viral) infectious diseases (which can be seen as including atherosclerosis), autoimmune diseases such as diabetes and its complications, (food) allergies such as asthma, rheumatism and Crohn’s disease, and cancer (2–10,90–109). Under Th1, zinc is closely involved in inhibiting tumour formation through suppressing angiogenesis near cancer cells (102–109). The tumour suppressor is endostatin, which is endogenous and zinc-dependent and is able to transform a deadly group of tumour cells into a harmless ‘dormant’ tumour whose growth is stunted by malnutrition as a result of no longer being able to promote angiogenesis (1–4,106). A sufficiency of Zn++ can therefore indirectly inhibit tumour growth by suppressing angiogenesis, but also directly via apoptosis as it does in prostate cancer cells by inactivating the enzyme 5-LO (104–106,109). It has already been shown above that Zn++ are able to inhibit the activity of this enzyme (93). Zinc deficiencies push the Th1/Th2 balance in the direction of Th2, partly by creating a shortage of NO and by raising LTB4 and PGE2 levels (2–10,15–19,26,27, 30–34d,38–42,45–70,92–94,97,98,100,101,103,109). The fact that, under Th2, shortages of NO develop as a result of zinc deficiency is also related to the fact that immune cells activated by antigens under Th1 release NO while under Th2 they do not (60). Moreover, an optimal NO level under Th1 brings the production of Il-2 and γ-interferon by the Th1 cells involved in immune activities to a timely end, which most likely reduces the danger of autoimmunity, as occurs in diabetes mellitus, multiple sclerosis (MS) and systemic lupus erythematosus (SLE) (2–6,29–40,51–54,60,64–68). SOME CONCLUSIONS A sufficiency of NO, zinc and other ENs can inhibit the proliferation of viruses such as HIV by inactivating the HIV protease and down-regulating both the production of LTB4 and the number of its BLTR receptors. In addition, adequate and efficient control of antigens such as those of HIV-1 requires powerful but time-limited immune activities under Th1. Shortages of ENs such as zinc © 1999 Harcourt Publishers Ltd

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promote the proliferation of Th2 cells at the expense of Th1 cells. Whether temporary, local, relative or absolute, zinc deficiencies help to shift the Th1/Th2 balance in favour of Th2 and thereby aid the proliferation of HIVs in Th2 cells. Deficiencies of ENs such as zinc prepare the ground for chronic (viral) infections that can result in diseases such as AIDS, atherosclerosis, autoimmune diseases, (food) allergies and cancers. Diabetes, as one of these autoimmune diseases, has received extra attention here but the prevention and treatment of cancer also has increasingly been shown to be reliant on an optimal provision of zinc. A typical Western diet, which contains a great deal – and in many cases to much – refined products such as sugars, fats and/or alcohol readily leads to a shortage of ENs. These shortages combined with an excess of fats have, as one consequence, an increased production of PGE2 and leukotrienes such as LTB4, as well as a reduced level of NO. These factors together result in more ROIs being brought into circulation, from which healthy cells need to be protected by precisely the antioxidants that have been put into short supply by this type of diet. An optimal diet should not contain too many calories. In addition, it should contain enough ENs per calorie consumed to allow diseases to be more effectively prevented and fought off by the undisturbed functioning of the most efficient immune activities. Increasingly, the need is not for more but for better nutrition. There are many indications that the food industry is beginning to direct its attention to this distinct gap in the market. With a scientifically responsible world-wide approach, it seems possible that epidemics such as AIDS could be brought under control in the foreseeable future. But it is not only parasitic organisms such as the AIDS virus that are constantly looking for a body whose immune system has become vulnerable. There are also the ‘diseases of civilization’ such as obesity, (food) allergies, autoimmune diseases, atherosclerosis and cancers that are following the same course and are taking an ever higher toll and for which the best and cheapest prevention or cure would seem to be an evolutionarily-determined optimal nutritional status and lifestyle (2–10). REFERENCES 1. Moran P. J., Welles S. L., Williams M. A. The inter-relation of maternal immune competence, HIV-1 viral load, and nutritional status in preventing vertical transmission: an alternative to chemoprophylaxis? Med Hypotheses 1998; 51: 389–397. 2. Sprietsma J. E. Zink, auto-immuunziekten, voedselallergieën en kanker. [Zinc, autoimmune diseases, food allergies and cancer] Uitgeverij Ankh-Hermes, Deventer (1988). 3. Sprietsma J. E. Overgewicht, hoge bloeddruk, diabetes mellitus, hart- en vaatziekten…een tekort aan spoor-elementen. [Obesity, hypertension, diabetes mellitus, atherosclerosis… a

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