Metallothionein

No treatise of trace elements would be complete without a discussion of binding proteins. The binding proteins for trace elements are of paramount importance for distribution to target sites of metabolism and excretion. These include a variety of proteins to include albumin, ceruloplasmin, a-glycoprotein, transferrin, and MT.

MTs, a family of low molecular weight-binding proteins (containing approximately 30% cysteine) for cadmium, copper, and zinc, are increased in several organs during the early phase of infection and are associated with redistribution of both essential and nonessential trace elements. This may be a normal response in common infections that could adversely influence the pathogenesis when the host is concomitantly exposed to potentially toxic trace elements, even at levels in the physiological range.94

Metalloenzymes have an important role in repair and regenerative processes in skin wounds. Demands for different enzymes vary according to the phase in the healing cascade and constituent events. Sequential changes in the concentrations of calcium, copper, magnesium, and zinc were studied in the incisional wound model in the rat over a 10 d period. Copper levels remained low (< 10 |j/g dry weight) throughout, but calcium, magnesium, and zinc increased from wounding, and peaked at about 5 d at a time of high inflammation, granulation tissue formation, and epidermal cell proliferation. Metal concentrations declined to normal by 7 d when inflammation had regressed, reepithelialization of the wound site was complete, and the "normalization" phase had commenced. Although the wound was overtly healed by day 10, the epidermis was still moderately hyperplastic. In view of competitive binding of trace metals at membrane receptors and carrier proteins, the ratio or balance between these trace metals was studied. Using immunocytochemistry, increases in MT immunoreactivity as an indication of zinc and copper activity were shown in the papillary dermis and in basal epidermal cells (near the wound margin 1 to 5 d after wounding). This is consistent with metalloenzyme requirements in inflammation and fibrogenesis. Calmodulin, a major cytosolic calcium-binding protein, was highest in maturing keratinocytes and in sebaceous gland cells of normal skin; it was notably more abundant in the epidermis near the wound margin and in reepithelializing areas at a time when local calcium levels were highest.95

Expression of the MT gene is upregulated in the skin following topical application of zinc and copper, and in wound margins, particularly in regions of high mitotic activity. This induction of MT in the wound margin may reflect its role in promoting cell proliferation and reepithelialization. The action of MTs in these processes may result from the large number of zinc-dependent and copper-dependent enzymes required for cell proliferation and matrix remodeling. In addition, selected growth factors may modulate MT gene expression and hence the ability of cells to proliferate.

MTs play a pivotal role in zinc-related cell homeostasis because of their high affinity for this trace element which is, in turn, relevant against oxidative stress and for the efficiency of the entire immune system, including natural killer (NK) cell activity. In order to accomplish this role, MTs sequester or dispense zinc during stress and inflammation to protect cells against reactive oxygen species. The gene expression of MT is affected by IL-6 for a prompt immune response. Concomitantly, MTs release zinc for the activity of antioxidant zinc-dependent enzymes, including poly(ADP-ribose)polymerase-1(PARP-1), which is involved in base excision DNA repair. This role of MTs is peculiar in young adult age during transient stress and inflammation, but not in aging, because stress-like and inflammatory conditions are persistent. This may lead MTs to turn off from a role of protection in young age to a deleterious one in aging, with subsequent appearance of age-related diseases (severe infections). The aim is to study the role played by MTs/IL-6/PARP-1 in the interplay on NK cell activity in the elderly population, in infected older patients (acute and remission phases by bronchopneumonia infection), and in healthy nonagenarian/centenarian subjects. MTmRNA (metallothionein messenger RNA) is high in lymphocytes from older people coupled with high IL-6, low zinc ion bioavail-ability, decreased NK cell activity, and impaired capacity of PARP-1 in base excision DNA repair. The same trend in this altered physiological cascade during aging also occurs in infected older patients (both acute and remission phases) with more marked immune damage, inflammatory condition, and very impaired PARP-1 in base excision DNA repair. By contrast, centenarian subjects display low MTmRNA, good zinc ion bioavailability, satisfactory NK cell activity, and higher capacity of PARP-1 in base excision DNA repair. These findings clearly demonstrate that the sequestering of zinc by MTs in aging is deleterious, because it leads to low zinc ion bioavailability with subsequent impairment of PARP-1 and NK cell activity and eventual appearance of severe infections. Physiological zinc supply (12 mg Zn(++)/d) for 1 month in the elderly and in infected older patients (remission phase) restores the activity of NK cells to values observed in healthy centenarians. Therefore, the zinc ion bioavailability by zinc-bound MT homeostasis is pivotal to reach healthy longevity and successfully age.96

Wound repair is initiated with the aggregation of platelets, formation of a fibrin clot, and release of growth factors from the activated coagulation pathways, injured cells, platelets, and extracellular matrix (ECM), followed by migration of inflammatory cells to the wound site. Thereafter, keratinocytes migrate over the wound, angiogenesis is initiated, and fibroblasts deposit and remodel the granulation tissue.

Cell migration, angiogenesis, degradation of provisional matrix, and remodeling of newly formed granulation tissue all require controlled degradation of the ECM. Disturbance in the balance between ECM production and degradation leads to formation of chronic ulcers with excessive ECM degradation, or to fibrosis, for example, hyper-trophic scars or keloids characterized by excessive accumulation of ECM components. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which as a group can degrade essentially all ECM components. To date, 20 members of the human MMP family have been identified. Based on their structure and substrate specificity, they can be divided into subgroups of collagenases, strome-lysins, stromelysin-like MMPs, gelatinases, membrane-type MMPs (MT-MMPs), and other MMPs.97

Trace Element Implications in Wound Repair

To reiterate, the normal wound repair process consists of three phases — inflammation, proliferation, and remodeling — that occur in a predictable series of cellular and biochemical events.

Growth factors play a role in cell division, migration, differentiation, protein expression, and enzyme production, and they have the potential to heal wounds by stimulating angiogenesis and cellular proliferation, affecting the production and the degradation of the extracellular matrix, and by being chemotactic for inflammatory cells and fibroblasts. There are seven major families of growth factors: epidermal growth factor (EGF), transforming growth factor-a (TGF-P), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), interleukins (ILs), and colony-stimulating factor (CSF).

Cytokines, especially interferon-a (IFN-a) and IFN-a-2b, may also reduce scar formation. These cytokines decrease the proliferation rate of fibroblasts and reduce the rate of collagen and fibronectin synthesis by reducing the production of mRNA. Nitric oxide synthase (NOS) and heat shock proteins (HSP) have an important role in wound healing, as do trace elements.

The normal healing response begins the moment the tissue is injured. As the blood components spill into the site of injury, the platelets come into contact with exposed collagen and other elements of the extracellular matrix. This contact triggers the platelets to release clotting factors as well as essential growth factors and cytokines, such as PDGF and TGF-p. Following hemostasis, the neutrophils enter the wound site and begin the critical task of phagocytosis to remove foreign materials, bacteria, and damaged tissue. As part of this inflammatory phase, the macrophages appear and continue the process of phagocytosis as well as release more PDGF and TGF-p. Once the wound site is cleaned out, fibroblasts migrate in to begin the proliferative phase and deposit new extracellular matrix. The new collagen matrix then becomes cross-linked and organized during the final remodeling phase. In order for this efficient and highly controlled repair process to take place, numerous cell-signaling events are required. In pathologic conditions, such as nonhealing pressure ulcers, this efficient and orderly process is lost, and the ulcers are locked into a state of chronic inflammation characterized by abundant neutrophil infiltration with associated ROS and destructive enzymes.

Healing proceeds only after the inflammation is controlled. On the opposite end of the spectrum, fibrosis is characterized by excessive matrix deposition and reduced remodeling. Often, fibrotic lesions are associated with increased densities of mast cells.98 Trace elements are intimately involved with all aspects of the healing spectrum.

Trace Element and Inflammatory Phase Implications

Highly potent substances are produced by the immune system. These substances include both cytokines and oxidant molecules, such as hydrogen peroxide, free radicals, and hypochlorous acid. The purpose of immune cell products is to destroy invading organisms and damaged tissue, bringing about recovery. However, oxidants and cytokines can damage healthy tissue. Excessive or inappropriate production of these substances is associated with mortality and morbidity after inflammation, infection, and trauma. Oxidants enhance IL-1, IL-8, and tumor necrosis factor (TNF) production in response to inflammatory stimuli by activating the nuclear transcription factor, NFkB. Sophisticated antioxidant defenses directly and indirectly protect the host against the damaging influence of cytokines and oxidants. Indirect protection is afforded by antioxidants, which reduce activation of NFkB, thereby preventing upregulation of cytokine production by oxidants. Cytokines increase both oxidant production and antioxidant defenses, thus minimizing damage to the host. Although antioxidant defenses interact when a component is compromised, the nature and extent of the defenses are influenced by dietary intake of sulfur amino acids, for glutathione synthesis, and vitamins E and C. In animal studies, in vivo and in vitro responses to inflammatory stimuli are influenced by dietary intake of copper, zinc, selenium, ^-acetylcysteine, cysteine, methionine, taurine, and vitamin E. Information from animal studies has yet to be fully translated into a clinical context. However, ^acetylcysteine, vitamin E, and a cocktail of antioxidant nutrients have reduced inflammatory symptoms in inflammatory joint disease, acute and chronic pancreatitis, and adult respiratory distress syndrome.99

It is widely appreciated that copper, iron, selenium, and zinc modulate immune function and influence the susceptibility of the host to infection. Nevertheless, the effect of individual trace elements or other micronutrients on components of innate immunity is difficult to design and interpret.

Zinc and iron concentrations in pus exceed normal serum. Calcium and magnesium levels in pus were two- to threefold lower and higher, respectively, than normal serum values. Lactoferrin concentrations were 880 ± 48 |Jg/ml, and ferritin levels were 20,726 ± 2667 ng/ml. Growth of an Escherichia coli strain was inhibited in pus at pH 5.5 but not at pH 7.4, and growth was enhanced by the addition of iron or zinc to E. coli suspended in pus at pH 6.7. Therefore, host defense mechanisms are enhanced by the restriction of the bioavailability of zinc and iron in suppurative infection.100 Zinc undernutrition results in lymphoid atrophy and reduced capacity to respond to many T-cell-dependent antigens. The plaque-forming cell response to heterologous erythrocytes is decreased, as is the function of B cells. In zinc-deficient rodents, the generation of cytotoxic lymphocytes in the spleen is reduced. Antibody-dependent cell-mediated cytotoxicity is largely unchanged. In acrodermatitis enteropathica, the lymphocyte proliferation response to mitogens is decreased, and there are significant changes in delayed hypersensitivity responses and in the proportion of various T-cell subsets. Neutrophil function is not changed by zinc deficiency. Iron deficiency results in a slight decrease in the number of rosette-forming T cells and a significant impairment of lymphocyte response to mitogens and antigens. Polymorphonuclear leukocytes are unable to kill ingested bacteria and fungi in an efficient manner. Copper deficiency impairs cell-mediated immunity, as does selenium deficiency, when it is associated with vitamin E deficiency.101

As a constituent of selenoproteins, selenium is needed for the proper functioning of neutrophils, macrophages, NK cells, T lymphocytes, and some other immune mechanisms. Elevated selenium intake may be associated with reduced cancer risk and may alleviate other pathological conditions, including oxidative stress and inflammation. Selenium appears to be a key nutrient in counteracting the development of virulence and inhibiting HIV progression to AIDS. It is required for sperm motility and may reduce the risk of miscarriage. Selenium deficiency has been linked to adverse mood states. Some findings suggest that selenium deficiency may be a risk factor in cardiovascular diseases.102

Lipopolysaccharide (LPS) produces ROS and NO in macrophages. These molecules are involved in inflammation associated with endotoxic shock. Selenium (Se), a biologically essential trace element, modulates the functions of many regulatory proteins involved in signal transduction and affects a variety of cellular activities, including cell growth and survival. Se attenuated LPS-induced ROS and NO production in murine macrophage cultures in vitro. This Se-decreased production of NO was demonstrated by decreases in both mRNA and protein expression for inducible NO synthase (iNOS). The preventive effects of Se on iNOS were p38 mitogen-activated protein kinase and NF-B dependent. Se specifically blocked the LPS-induced activation of p38 but not that of c-jun-N-terminal kinase and extracellular signal-regulated kinase; the p38-specific pathway was confirmed using p38 inhibitor SB 203580.

These results suggest the mechanism by which Se may act as an anti-inflammatory agent and that Se may be considered as a possible preventive intervention for endot-oxemia, particularly in Se-deficient geographical locations.103 Long-term intake greater than 0.4 mg/d Se in adults can produce adverse effects. In burn patients, balance studies have shown the need for 200 mcg Se per day for 2 weeks and then 60 mcg/d.104

Again, selenium represents a trace element foundin antioxidant enzyme glu-tathion-peroxidase. Its anti-inflammatory activity is based on the elimination of hydroperoxides produced in the site of inflammation.105

Patients with systemic inflammatory response syndrome (SIRS) and sepsis exhibit decreased plasma selenium and glutathione peroxidase activity. Moreover, the degree of selenium deficiency correlates with the severity of the disease and the incidence of mortality. Patients with SIRS and sepsis are exposed to severe oxidative stress. Selenoenzymes play a major role in protecting cells against peroxidation, especially lipid peroxidation, and are involved in the regulation of inflammatory processes.106

In bacterial infections, septicemia, pneumonia, erysipelas, and meningitis, the plasma concentrations of selenium, iron, and zinc are decreased. Plasma copper was unchanged in patients with erysipelas but increased in other types of bacterial infections. Although the patients with viral infections showed similar shifts of the trace elements as were observed in patients with bacterial infections, the changes were not as pronounced. A low plasma selenium level less than 0.8 mumol/l was found in only 6% of the patients with viral infections in contrast to 63% of the patients with septicemia or 57% of the patients with pneumonia. Furthermore, in viral infections 60% of the zinc values were below the mean level of 12.8 mumol/l observed in healthy controls as compared with 90% of the values in patients with sepsis or 92% of the values in patients with pneumonia. The onset of change in trace elements occurred within a few days and persisted for several weeks. These changes seem to be nonspecific and are independent of the agent causing infection. The different types of infections were followed by changes in most of the plasma proteins, which are known to be associated with an inflammatory reaction. The changes in plasma proteins were most pronounced in patients with sepsis and pneumonia. Therefore, patients with sepsis having a high degree of inflammation did not show a positive correlation between the severity of the disease — as judged by plasma proteins — and the alterations of trace elements.107

The addition of copper and zinc salts to human peripheral blood leukocytes cultured in complete medium containing endotoxin and fetal calf serum stimulated tumor necrosis factor (TNF) secretion in a concentration-dependent manner. The secretion of interleukin-1p (IL-1P) and interleukin-6 (IL-6) was inhibited by copper under the same culture conditions, although zinc stimulated IL-ip secretion in a concentration-dependent manner and had no effect on leukocyte IL-6 release. Both copper and zinc induced increases in TNF mRNA (54 and 14%, respectively) when compared to cells cultured in complete medium alone. In serum-free, low endotoxin medium (less than 6 pg/ml), both copper and zinc failed to stimulate either TNF or IL-ip secretion. Under the same conditions, the addition of lipopolysaccharide (LPS), at concentrations above 0.01 |g/ml, induced a concentration-dependent release of both cytokines. When either copper or zinc was combined with 0.01 |g/ml LPS, a synergistic stimulation of TNF secretion resulted. IL-1p secretion, unlike TNF, was not synergistically stimulated by combining metals and LPS in serum-free medium. Combining copper and zinc with inhibitors of TNF secretion, TGF-P, prostaglandin E2, and plasma a-globulins, resulted in a reduction of the suppressive effects of each of these agents. Thus, trace metals copper and zinc may play important and possibly distinct roles in regulating leukocyte secretion of TNF, IL-1P, and IL-6.108

The fall in serum iron and zinc and the rise in serum copper in an acute phase response are brought about by changes in the concentration of specific tissue proteins controlled by cytokines, especially IL-1, TNF, and IL-6. These are generally believed to be beneficial aspects of the early acute phase response. One difficulty associated with these changes is that assessment of status for these elements is particularly difficult, because plasma concentration may bear little relationship to tissue status. Simultaneous assessment of the acute phase response — for example, serum C-reactive protein — together with trace elements and the monitoring of changes in concentrations may, however, permit interpretation of trace element requirements. Suggestions are made for the requirements for these and other essential elements during enteral or intravenous nutrition, together with proposed methods of interpreting laboratory tests.109

Zinc induces monokine secretion by monocytes; however, effects of zinc on T cells appear contradictory. Apart from enhanced lymphocyte proliferation in peripheral blood mononuclear cells (PBMCs), inhibitory properties of high zinc dosages have also been described. PBMCs failed to produce lymphokines like IFN-y after stimulation with zinc in a serum- and LPS-free cell culture system, whereas monokine secretion (IL-1P) has occurred. Zinc-uptake studies with the zinc-specific fluorescent probe zinquin revealed that zinc is taken up by PBMCs within a few minutes, reaching nearly equal levels in PBMCs, isolated monocytes, and T cells. However, if zinc was depleted 1 h after monocyte induction, zinc-free precultured T cells were stimulated to secrete IFN-y by zinc-induced monokines. Furthermore, the necessity for a cell-cell interaction between monocytes and T cells for IFN-y induction has been elucidated. Zinc ions inhibited the proliferation of the IL-1-dependent T cell line D 10N in a dose-dependent manner, suggesting a direct inhibitory effect of zinc. By immunoprecipitation, a specific inhibition of IL-1 receptor-associated protein kinase (IRAK) by zinc ions was shown. Therefore, in contrast to an indirect stimulation of T cells due to zinc-induced mono-kines, higher concentrations of zinc directly inhibit T cell functions by means of specific inhibition of IRAK and subsequent signaling events, such as NFkB activation. The divergent effects of zinc on different cell populations, depending on the zinc concentration, could explain contradictory results of zinc stimulation.110

Zinc deficiency can change immune functions prematurely from predominantly cellular T helper cell (Th1) responses to humoral Th2 responses. Th1 cells produce cytokines such as IL-2 and IFN-y, thereby controlling viral infections and other intracellular pathogens more effectively than Th2 responses through cytokines such as IL-4, IL-5, IL-6, and IL-10. The accelerated shift from the production of extra Th1 cells during these cellular immune activities to more Th2 cells with their predominantly humoral immune functions, caused by such a zinc deficiency, adversely influences the course of diseases such as leprosy, schistosomiasis, leish-maniasis, and AIDS, and can result in allergies. It is noteworthy that AIDS viruses (HIVs) do not replicate in Th1 cells, which probably contain more zinc, but preferentially in the Th0 and Th2 cells; all the more so because zinc and copper ions are known to inhibit intracellular HIV replication. Considering the above Th1/Th2 switch, real prospects seem to be offered of vaccination against such parasites as leishmania and against HIVs.111

The nutritional status and needs of older people are associated with age-related biological and often socioeconomic changes. Decreased food intake, a sedentary lifestyle, and reduced energy expenditure in older adults become critical risk factors for malnutrition, especially protein and micronutrients. Surveys indicate that older people are particularly at risk for marginal deficiency of vitamins and trace ele-ments.112 This may explain why aging is associated with impaired immune responses and increased infection-related morbidity.113

The T lymphocyte is more susceptible to aging than the B lymphocyte, for which in vitro functions are almost preserved. All functions of cell-mediated immunity are decreased in older adults. T lymphocytes are less mature and present decreased T helper and T suppressive functions, decreased T proliferative ability responses to stimuli, and lower interleukin synthesis. T-dependent antibody responses are also decreased: primary responses are diminished, and secondary responses are less specific. Nevertheless, in healthy older adults such immune deficiency remains minor. When undernutrition occurs in aged individuals, a profound immunodeficit rapidly follows. Prevention of prompt treatment of all undernutrition states is a major concern in geriatrics.114

Trace Element and Proliferation Phase Implications

Numerous studies report the role of interactions between keratinocytes and fibro-blasts, but the relationship between wound healing myofibroblasts and keratinocytes is not clear, even though these two cell types coexist during healing. To determine the speed and quality of epithelialization, the influence of myofibroblasts on kerat-inocyte growth and differentiation was studied using an in vitro skin model. When the dermis was populated with fibroblasts, a continuous epidermis was formed in 7 to 10 d. In contrast, with wound healing myofibroblasts or without cell in dermis, the complete reepithelialization never occurred over the 10 d period studied. After 7 further days of epidermal differentiation, histology showed a more disorganized epidermis, and the expression of basement membrane constituents was reduced when wound healing myofibroblasts or no cells were added in the dermis instead of fibroblasts. Results suggest that wound healing myofibroblasts are not efficient to stimulate keratinocyte growth and differentiation. Treatment of fibroblasts with TGF-P1 induced an increase of epidermal cell differentiation as seen when myofibroblasts were present. However, this cytokine did not change the reepithelialization rate and induced an increase of basement membrane matrix deposition in opposition to myofibroblasts. Thus TGF-P1 action is not sufficient to explain all the different keratinocyte reactions toward fibroblasts and wound healing myofibroblasts. Myo-fibroblasts, in contrast to fibroblasts, seem to have a limited role in the reepithelial-ization process and might be more associated with the increased extracellular matrix secretion.115

Some integrins that are modulated by trace elements and expressed by basal layer keratinocytes play an essential part in healing, notably a2p1, a3p1, a6p4, and aVp5, whose expression and distribution in epidermis are modified during the reepithelialization phase. Integrin expression was studied in proliferating kerati-nocytes in monolayer cultures and in reconstituted skin that included a differentiation state. The expression of a6 was induced by zinc, copper, and manganese. The inductive effect of zinc was particularly notable on integrins affecting cellular mobility in the proliferation phase of wound healing (a3, a6, aV) and that of copper on integrins expressed by suprabasally differentiated keratinocytes during the final healing phase (a2, p1, and a6), but manganese had a mixed effect.116

Selenium is an essential trace element, and it is well known that selenium is necessary for cell culture. However, the mechanism underlying the role of selenium in cellular proliferation and survival is still unknown. Investigators using Jurkat cells showed that selenium deficiency in a serum-free medium decreased the selenium-dependent enzyme activity (glutathione peroxidases and thioredoxin reductase) within cells and cell viability. To understand the mechanism of this effect of selenium, the effect of other antioxidants, the act by different mechanisms was examined. Vitamin E, a lipid-soluble radical-scavenging antioxidant, completely blocked sele nium deficiency-induced cell death, although a-tocopherol (biologically the most active form of vitamin E) could not preserve selenium-dependent enzyme activity. Other antioxidants, such as different isoforms and derivatives of vitamin E, BO-653, and deferoxamine mesylate, also exerted an inhibitory effect. However, the water-soluble antioxidants, such as ascorbic acid, N-acetyl cysteine, and glutathione, displayed no such effect. Dichlorodihydrofluorescein (DCF) assay revealed that cellular ROS increased before cell death, and sodium selenite and -tocopherol inhibited ROS increase in a dose-dependent manner. The generation of lipid hydroperoxides was observed by fluorescence probe diphenyl-1-pyrenylphosphine (DPPP) and highperformance liquid chromatography (HPLC) chemiluminescence only in selenium-deficient cells. These results suggest that the ROS, especially lipid hydroperoxides, are involved in the cell death caused by selenium deficiency and that selenium and vitamin E cooperate in the defense against oxidative stress upon cells by detoxifying and inhibiting the formation of lipid hydroperoxides.117

Zinc is not only an important nutrient and cofactor of numerous enzymes and transcription factors, but it also acts as an intracellular mediator, similarly to calcium. The recent discovery of its intracellular molecular pathways opens the door to new fields of drug design. Zinc homeostasis results from a coordinated regulation by different proteins involved in uptake excretion and intracellular storage/trafficking of zinc. These proteins are membranous transporters, belonging to the ZIP and ZnT families, and MTs. Their principal function is to provide zinc to new synthesized proteins important for several functions such as gene expression, immunity, reproduction, or protection against free radicals damage. Zinc intracellular concentration is correlated to cell fate (i.e., proliferation, differentiation, or apoptosis), and modifications of zinc homeostasis are observed in several pathologies affecting humans at any stage of life. Two zinc-related diseases, acrodermatitis enteropathica and lethal milk syndrome, have recently been related to mutations in zinc transporters SLC39A4 and ZnT-4, respectively. Zinc acts as an inhibitor of apoptosis, while its depletion induces programmed cell death in many cell lines. However, excess zinc can also be cytotoxic, and zinc transporters as well as MTs serve as zinc detoxification systems. Several zinc channels controlling the intracellular zinc movements and the free form of the metal maintain the intracellular zinc homeostasis and thus the balance between life and cell death. Apart from these general activities, zinc has particular biological roles in some specialized cells. It acts as a paracrine regulator in pancreatic cell, neuron, or neutrophil activity by a mechanism of vesicles-mediated metal excretion and uptake.118

Keratinocyte growth factor (KGF) stimulates epithelial cell differentiation and proliferation in animals and promises to be of major importance for wound healing. Local protein administration, however, has been shown to be ineffective due to enzymes and proteases in the wound fluid. Liposomes containing the KGF cDNA gene constructs were effective in improving epidermal and dermal regeneration. KGF gene transfer to acute wounds may represent a new therapeutic strategy to enhance wound healing.119

Finally, proliferation of keratinocytes has been demonstrated in toxic epidermal necrolysis, a rare disease observed as a consequence of adverse reactions to drugs. yIFN-y, soluble aTNF-a, and soluble Fas ligand (sFas-L) are present in much higher concentrations in the blister fluids of patients with toxic epidermal necrolysis (TEN). IFN-y and to a lesser extent IL-18 were produced by mononuclear cells present in the fluid. Other cytokines (TNF-a, sFas-L, IL-10) originated from activated kerati-nocytes. Fas-L was overexpressed on the membranes of keratinocytes in lesional skin in situ. The Th1 profile of T-lymphocyte activation found in the blister fluid of patients with TEN is consistent with a key role for drug-specific cytotoxic T lymphocytes (CTL) as previously reported — the activation of keratinocytes by IFN-y, making them sensitive to cell-mediated cytolysis. It was proposed that the production of Fas-L, TNF-a, and IL-10 by keratinocytes could be a defense mechanism against CTL rather than a way of propagating apoptosis among epidermal cells.120

Thus, keratinocyte proliferation and migration are essential for the reconstruction of the cutaneous barrier after skin injury. Interestingly, thermal waters rich in trace elements (e.g., boron and manganese) are known to be able to improve wound healing. In order to understand the mechanism of action of this effect, investigators have studied the in vitro modulation of keratinocyte migration and proliferation by boron and manganese salts, which are present in high concentrations in thermal water (Saint Gervais). In vitro study has demonstrated that incubating keratinocytes for 24 h with boron salts at concentrations between 0.5 and 10 mcg/ml or manganese salts at concentrations between 0.1 and 1.5 mcg/ml accelerated wound closure compared with control medium (+20%). As this acceleration was not related to an increase in keratinocyte proliferation, it was suggested that boron and manganese act on wound healing mainly by increasing the migration of keratinocytes.121

Trace Element and Remodeling Phase Implications

Wound healing encompasses coagulation, inflammation, angiogenesis, fibroplasia, contraction, epithelialization, and remodeling. Granulation tissue is produced following incision of tissue, such as skin, abdominal wall, or the gastrointestinal tract, and the strength of the wound is determined primarily by the collagen content early in the healing course.122

Zinc (Zn) retains insulin-like growth factor binding proteins (IGFBPs) on the surface of cultured cells, lowers the affinity of cell-associated IGFBPs, and increases the affinity of the cell surface insulin-like growth factor (IGF)-type 1 receptor (IGF-1R). Investigators have characterized a mechanism by which the trace micronutrient Zn could regulate IGF activity.123 Another study showed that zinc decreases IGF binding to fibroblast-associated IGFBPs by lowering the affinity of the IGF-IGFBP interaction.124

The addition of copper and zinc salts to human peripheral blood leukocytes cultured in complete medium containing endotoxin and fetal calf serum stimulated TNF secretion in a concentration-dependent manner. The secretion of IL-1P (IL-1P) and IL-6 was inhibited by copper under the same culture conditions, while zinc stimulated IL-1P secretion in a concentration-dependent manner and had no effect on leukocyte IL-6 release. Both copper and zinc induced increases in TNF mRNA (54 and 14%, respectively) when compared to cells cultured in complete medium alone. In serum-free, low endotoxin medium (less than 6 pg/ml), both copper and zinc failed to stimulate either TNF or IL-1P secretion. Under the same conditions, the addition of lipopolysaccharide (LPS) at concentrations above 0.01 |g/ml induced a concentration-dependent release of both cytokines. When either copper or zinc was combined with 0.01 |g/ml LPS, a synergistic stimulation of TNF secretion resulted. IL-1P secretion, unlike TNF, was not synergistically stimulated by combining metals and LPS in serum-free medium. Combining copper and zinc with inhibitors of TNF secretion, TGF-P, prostaglandin E2, and plasma a-globulins resulted in a reduction of the suppressive effects of each of these agents. This suggests that the trace metals copper and zinc may play important and possibly distinct roles in regulating leukocyte secretion of TNF, IL-1P, and IL-6.125

To evaluate the modulatory effects of trace metals on lymphocyte growth and maturation, thymidine uptake (TU), protein, ATP, Fe, Cu, Zn, ferritin, CD3, CD4, CD8 antigens, surface transferrin receptors (TFR), and interleukin-2 receptors (IL-2R) were assessed in normal and T-cell leukemia human lymphocytes (ALL), cultured in media with varying Fe, Cu, and Zn concentrations [Me]. The response of normal lymphocytes to stimuli is sensitive to variation in trace metals, whereas this response, absent in ALL lymphocytes, reappears only in media with low [Me] and is independent from TFR.126

Zinc induces monocytes to produce IL-1, IL-6, and TNF-a in peripheral blood mononuclear cells and separated monocytes. This effect is higher in serum-free medium. However, only in the presence of serum does zinc also induce T cells to produce lymphokines. This effect on T cells is mediated by cytokines produced by monocytes. Stimulation also requires cell-to-cell contact of monocytes and T cells.127 INF-a and INF-a 2P may also reduce scar formation. These cytokines decrease the proliferation rate of fibroblasts.128 Zinc is an essential trace element that also increases osteoblast numbers and bone formation.129 In addition, other trace elements play important roles in regulating the metabolism of the extracellular matrix.130

Free radicals prevent wound healing. Findings suggest that collagen is denatured by scavenging the hydroxyl radical before fibroblasts are damaged, so that the radical may influence the remodeling of collagen.131

The state of excessive fibroblastic proliferation for wound healing results in hypertrophic and keloid scars. The levels of zinc (Zn), copper (Cu), manganese (Mn), and selenium (Se) in serum, normal skin, and scar tissue of 40 patients with keloid and hypertrophic scars were assessed. There was a significant increase in manganese (Mn) levels in the skin of patients with burns, trauma, and surgical incisions compared to controls. Furthermore, the zinc, copper, and selenium contents of the skin in patients with incisions were decreased significantly when compared to other groups. No significant changes occurred regarding serum levels of zinc, copper, manganese, and selenium in the different groups.132

In a study aimed at testing human skin wound healing improvement with a 21 d supplementation of 1 g ascorbic acid (AA) and 0.2 g pantothenic acid (PA), 49 patients undergoing surgery for tattoos by the successive resections procedure entered a double-blind, prospective, and randomized study. Tests performed on both skin and scars determined hydroxyproline concentrations, number of fibroblasts, trace element contents, and mechanical properties. In the 18 patients who received supplementation, it was shown that in skin (day 8), Fe increased (p < 0.05) and Mn decreased (p < 0.05); in scars (day 21), Cu (p = 0.07) and Mn (p < 0.01) decreased, and Mg (p < 0.05) increased; the mechanical properties of scars in group A were significantly correlated to their contents in Fe, Cu, and Zn, whereas no correlation was shown in group B. In blood, AA increased after surgery with supplementation, whereas it decreased in controls. Although no major improvement of the wound healing process could be documented in this study, results suggested that the benefit of AA and PA supplementation could be due to the variations of the trace elements, as they are correlated to mechanical properties of the scars.133

Normal wound healing is a complex, highly regulated dynamic process that requires coordinate responses of both epidermal and dermal compartments. To accomplish the healing process, several growth factors, chemokines, and matrix elements signal both cell proliferation and migration during the inflammatory and reparative phases and limit these responses during the remodeling phase. A stimulated keratinocyte/target cell coculture system revealed that IFN-y inducible protein 9 acts as a soluble keratinocyte-derived paracrine factor for both fibroblasts and keratinocytes. Further, it was found that in both fibroblasts and undifferentiated keratinocytes, IFN-y inducible protein 9 exerted its action through modulation of a cytosolic protease, calpain. Interestingly, IFN-y inducible protein 9 increased calpain activity in undifferentiated keratinocytes, whereas the same chemokine inhibited the calpain activity in fibroblasts. This provides for a model whereby redifferentiated basal keratinocytes could limit fibroblast repopulation of the dermis underlying healed wounds while simultaneously promoting reepithelialization of the remaining provisional wound.134

Finally, the interaction of other micronutrients such as electrolytes with the actions of trace elements may impact on successful wound healing. Signal trans-duction of many intracellular events is initiated by a minute influx of calcium ions into the cells, resulting in the formation of a calcium-calmodulin complex and cAMP. There is reciprocity between epidermal calmodulin and cAMP levels, which may be modulated by external factors such as zinc.135

Recommendations for Supplementation and Avoiding Toxicity

Although trace elements are micronutrients, there are major roles in wound healing that are carried out with adequate supplementation. Dosing for wound healing will be contingent on sampling fluid/tissue, comorbidity, salt, bioabsorption to target sites, and current/historical intake/excretion. To provide the recommended daily dietary allowances, 1 to 1.5 l of most commercially available enteral formulas will be needed. Injectable products will be dosed lower than enteral products because most products will be given intravenously, which will provide enhanced bioavail-ability. Deficiencies are best determined by recognition, then supplementation, and subsequent serial monitoring of signs and symptoms. From a pragmatic perspective, chromium (Cr), copper (Cu), iron (Fe), selenium (Se), and zinc (Zn) inadequacies are likely respectively manifested by hyperglycemia (Cr), neutropenia (associated with microcytic/hypochromic anemia), as well as kinky hair (Cu), microcytic/hypo-chromic anemia (Fe), muscle pain/cardiomyopathy (Se), and diarrhea/alopecia/delayed wound healing (Zn) phenomena.

Similarly, signs and symptoms are often associated with micronutrient overdosing. Chromium, copper, iron, selenium, and zinc toxicities are likely respectively manifested by hypoglycemia (Cr), Wilson's Disease (Cu), hyperferritinemia (Fe), dermatitis/garlic breath (Se), and hyperamylasemia (Zn).

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