Cytokines, Inflammation and Pain

Darned microbial litterbugs…8)

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Cytokines are small secreted proteins released by cells have a specific effect on the interactions and communications between cells. Cytokine is a general name; other names include lymphokine (cytokines made by lymphocytes), monokine (cytokines made by monocytes), chemokine (cytokines with chemotactic activities), and interleukin (cytokines made by one leukocyte and acting on other leukocytes). Cytokines may act on the cells that secrete them (autocrine action), on nearby cells (paracrine action), or in some instances on distant cells (endocrine action). There are both pro-inflammatory cytokines and anti-inflammatory cytokines. There is significant evidence showing that certain cytokines/chemokines are involved in not only the initiation but also the persistence of pathologic pain by directly activating nociceptive sensory neurons. Certain inflammatory cytokines are also involved in nerve-injury/inflammation-induced central sensitization, and are related to the development of contralateral hyperalgesia/allodynia. The discussion presented in this chapter describes several key pro-inflammatory cytokines/chemokines and anti-inflammatory cytokines, their relation with pathological pain in animals and human patients, and possible underlying mechanisms.

Keywords: cytokine, inflammation, pain, hyperalgesia


Inflammatory responses in the peripheral and central nervous systems play key roles in the development and persistence of many pathological pain states []. Certain inflammatory cytokines in spinal cord, dorsal root ganglion (DRG), injured nerve or skin are known to be associated with pain behaviors and with the generation of abnormal spontaneous activity from injured nerve fibers or compressed/inflamed DRG neurons.

Cytokines are small secreted proteins released by cells have a specific effect on the interactions and communications between cells. Cytokine is a general name; other names include lymphokine (cytokines made by lymphocytes), monokine (cytokines made by monocytes), chemokine (cytokines with chemotactic activities), and interleukin (cytokines made by one leukocyte and acting on other leukocytes). Cytokines may act on the cells that secrete them (autocrine action), on nearby cells (paracrine action), or in some instances on distant cells (endocrine action).

It is common for different cell types to secrete the same cytokine or for a single cytokine to act on several different cell types (pleiotropy). Cytokines are redundant in their activity, meaning similar functions can be stimulated by different cytokines. They are often produced in a cascade, as one cytokine stimulates its target cells to make additional cytokines. Cytokines can also act synergistically or antagonistically.

Cytokines are made by many cell populations, but the predominant producers are helper T cells (Th) and macrophages. Cytokines may be produced in and by peripheral nerve tissue during physiological and pathological processes by resident and recruited macrophages, mast cells, endothelial cells, and Schwann cells. Following a peripheral nerve injury, macrophages and Schwann cells that gather around the injured site of the nerve secrete cytokines and specific growth factors required for nerve regeneration. Localized inflammatory irritation of the dorsal root ganglion (DRG) not only increases pro-inflammatory cytokines but also decreases anti-inflammatory cytokines []. Cytokines can also be synthesized and released from the herniated nucleus pulposus, synthesized inside the spinal cord [], the DRG soma [], or the inflamed skin []. Furthermore, cytokines may be transported in a retrograde fashion from the periphery, via axonal or non-axonal mechanisms, to the DRG and dorsal horn, where they can have profound effects on neuronal activity [] and therefore contribute to the etiology of various pathological pain states.

2. Cytokines and Pain


Proinflammatory cytokines are produced predominantly by activated macrophages and are involved in the up-regulation of inflammatory reactions. There is abundant evidence that certain pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α are involved in the process of pathological pain.

IL-1β is released primarily by monocytes and macrophages as well as by nonimmune cells, such as fibroblasts and endothelial cells, during cell injury, infection, invasion, and inflammation. Very recently, it was found that IL-1β is expressed in nociceptive DRG neurons []. IL-1β expression is enhanced following crush injury to peripheral nerve and after trauma in microglia and astrocytes in the central nervous system (CNS) []. IL-1β can produce hyperalgesia following either intraperitoneal, intracerebroventricular or intraplantar injection []. Moreover, IL-1β was found to increase the production of substance P and prostaglandin E2 (PGE2) in a number of neuronal and glial cells []. IL-1ra, a specific IL-1 receptor antagonist, competitively binds to the same receptor as IL-1β but does not transduce a cellular signal, thereby blocking IL-1β-mediated cellular changes. Administrations of IL-1ra and other anti-inflammatory cytokines have been demonstrated to prevent or attenuate cytokine-mediated inflammatory hyperalgesia [] and nerve-injury induced mechanical allodynia [].

IL-6 has been shown to play a central role in the neuronal reaction to nerve injury. Suppression of IL-6R by in vivo application of anti-IL-6R antibodies led to reduced regenerative effects []. IL-6 is also involved in microglial and astrocytic activation as well as in regulation of neuronal neuropeptides expression []. There is evidence that IL-6 contributes to the development of neuropathic pain behavior following a peripheral nerve injury []. For example, sciatic cryoneurolysis, a sympathetically-independent model of neuropathic pain involving repeatedly freezing and thawing a section of the sciatic nerve, results in increased IL-6 immunoreactivity in the spinal cord []. In addition, intrathecal infusion of IL-6 induces tactile allodynia and thermal hyperalgesia in intact and nerve-injured rats, respectively.

TNF-α, also known as cachectin, is another inflammatory cytokine that plays a well-established, key role in some pain models. TNF acts on several different signaling pathways through two cell surface receptors, TNFR1 and TNFR2 to regulate apoptotic pathways, NF-kB activation of inflammation, and activate stress-activated protein kinases (SAPKs). TNF-α receptors are present in both neurons and glia []. TNF-α has been shown to play important roles in both inflammatory and neuropathic hyperalgesia. Intraplantar injection of complete Freund’s adjuvant in adult rats resulted in significant elevation in the levels of TNF-α, IL-1β, and nerve growth factor (NGF) in the inflamed paw. A single injection of anti-TNF-α antiserum before the CFA significantly delayed the onset of the resultant inflammatory hyperalgesia and reduced IL-1β but not NGF levels []. Intraplantar injection of TNF-α also produces mechanical [] and thermal hyperalgesia []. It has been found that TNF-α injected into nerves induces Wallerian degeneration [] and generates the transient display of behaviors and endoneurial pathologies found in experimentally painful nerve injury []. TNF binding protein (TNF-BP), an inhibitor of TNF, is a soluble form of a transmembrane TNF-receptor. When TNF-BP is administered systemically, the hyperalgesia normally observed after lipopolysaccharide (LPS) administration is completely eliminated []. Intrathecal administration of a combination of TNF-BP and IL-1 antagonist attenuated mechanical allodynia in rats with L5 spinal nerve transection [].


A variety of cytokines are known to induce chemotaxis. One particular subgroup of structurally related cytokines is known as chemokines. The term chemotactic cytokines (CHEMOtactic CytoKINES) usually refers to this. These factors represent a family of low molecular weight secreted proteins that primarily function in the activation and migration of leukocytes although some of them also possess a variety of other functions. Chemokines have conserved cysteine residues that allow them to be assigned to four groups: C-C chemokines (RANTES, monocyte chemoattractant protein or MCP-1, monocyte inflammatory protein or MIP-1α, and MIP-1β), C-X-C chemokines (IL-8 also called growth related oncogene or GRO/KC), C chemokines (lymphotactin), and CXXXC chemokines (fractalkine).

Various chemokines including MIP-1α, MCP-1 and GRO/KC are up-regulated not only in models of neuroinflammatory [] and demylinating diseases, but also in various forms of CNS trauma [] and in injured peripheral nerve []. Receptors for MCP-1, MIP-1α and GRO/KC are expressed on DRG neurons []. Interestingly, mice lacking the CCR2 receptor completely fail to develop mechanical allodynia in the partial sciatic injury model although pain sensitivity in uninjured animals is normal. In the same study, normal mice showed a sustained upregulation of the receptors in both DRG and peripheral nerve after the injury []. This suggests that the chemokines, including MCP-1 in particular, play very key roles in neuropathic pain as well as in neuroinflammatory conditions.


The anti-inflammatory cytokines are a series of immunoregulatory molecules that control the pro-inflammatory cytokine response. Cytokines act in concert with specific cytokine inhibitors and soluble cytokine receptors to regulate the human immune response. Their physiologic role in inflammation and pathologic role in systemic inflammatory states are increasingly recognized. Major anti-inflammatory cytokines include interleukin (IL)-1 receptor antagonist, IL-4, IL-10, IL-11, and IL-13. Leukemia inhibitory factor, interferon-alpha, IL-6, and transforming growth factor (TGF)-β are categorized as either anti-inflammatory or pro-inflammatory cytokines, under various circumstances. Specific cytokine receptors for IL-1, TNF-α, and IL-18 also function as inhibitors for pro-inflammatory cytokines.

Among all the anti-inflammatory cytokines, IL-10 is a cytokine with potent anti-inflammatory properties, repressing the expression of inflammatory cytokines such as TNF-α, IL-6 and IL-1 by activated macrophages. In addition, IL-10 can up-regulate endogenous anti-cytokines and down-regulate pro-inflammatory cytokine receptors. Thus, it can counter-regulate production and function of pro-inflammatory cytokines at multiple levels. Acute administration of IL-10 protein has been well-documented to suppress the development of spinally-mediated pain facilitation in diverse animal models such as peripheral neuritis, spinal cord excitotoxic injury, and peripheral nerve injury []. Blocking spinal IL-10, on the other hand, has been found to prevent and even reverse established neuropathic pain behaviors []. Recent clinical studies also indicate that low blood levels of IL-10 and another anti-inflammatory cytokine, IL-4, could be key to chronic pain since low concentrations of these two cytokines were found in patients with chronic widespread pain [].

The family of TGF-β comprises 5 different isoforms (TGF-β1 to -β5). TGF-β1 is found in meninges, choroid plexus, and peripheral ganglia and nerves []. It is known that TGF-β suppresses cytokine production by inhibiting macrophage and Th1 cell activity; counteracts IL-1, IL-2, IL-6, and TNF; and induces IL-1ra 6 []. Its mRNA is induced following axotomy and may be involved in a negative-feedback loop to limit the extent of glial activation []. TGF-β1 also antagonizes nitric oxide production in macrophages []. Nitric oxide has been strongly implicated in the final common pathway of neuropathic pain []. It is expected that by its anti-cytokine action, TGF-β1 or agents that induce its activity may be effective therapy for neuropathic pain.


In the CNS, there are two types of glial cells, microglia and astrocytes, which can be activated by excitatory neurotransmitters released from nearby neurons. These neurotransmitters include EAA, SP, PGEs, adenosine triphosphate (ATP), and nitric oxide. A novel neuron to glia signal is fractalkine, a protein expressed on the extracellular surface of neurons []. Fractalkine is tethered to the neuronal membrane by a mucin stalk. When the neuron is sufficiently activated, the stalk breaks, releasing fractalkine into the extracellular fluid. As immunocompetent cells, activated glia release several key pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 [].

It has been well demonstrated that spinal glial activation is necessary for induction of the neuropathic pain state []. Spinal administration of glial activator, fractalkine, induces cutaneous hyperalgesia, whereas spinal administration of a fractalkine receptor antagonist blocks neuropathic pain []. Furthermore, blocking the activation of spinal cord glia with the inhibitor fluorocitrate blocks the pathological pain state in rats with peripheral sciatic nerve neuritis []. Recently, it was found that administration of a new glia-specific inhibitor, minocycline, blocked the development of neuropathic pain. Minocycline, a lipid-soluble tetracycline derivative with anti-inflammatory effects, inhibits an IL-1β-converting enzyme and inducible nitric oxide synthesis up-regulation. Minocycline also prevents glial cell proliferation and inhibits the activation of p38 MAPK [].

Non-neuronal cells in the peripheral nervous system also react to nerve injury. In addition to hematogenous macrophage infiltration, the satellite glia that surround the somata of sensory neurons proliferate [], elaborate processes [], and become immunoreactive for glial fibrillary acidic protein (GFAP) [].


There is evidence that pro-inflammatory cytokines (e.g., IL-1β, TNF-α) [] and chemokines (e.g., MCP-1) [] may directly modulate neuronal activity in various classes of neurons in the peripheral and central nervous system. In the peripheral nervous system, abnormal spontaneous activity can be evoked from nociceptive neurons by topical application of TNF-α to the peripheral axons in vivo [], or to the somata of the DRG neurons in vitro []. Large, myelinated fast conducting Aβ neurons can also be excited by topical application of TNF-α to the DRG [] or by an autologous HNP extract []. TNF-α can enhance the sensitivity of sensory neurons to the excitation produced by capsaicin and this enhancement likely is mediated by the neuronal production of prostaglandins []. It was found that TNF-α-induced neuronal excitation is mediated by cAMP-dependent protein kinase (PKA) pathway []. The p38 mitogen-activated protein kinase (MAPK) is also involved in TNF-α-induced cutaneous hypersensitivity to mechanical or thermal stimulation []. Results obtained from IL-6 knockout mice indicates that IL-6 plays a facilitating role in sympathetic sprouting induced by nerve injury and that its effect on pain behavior is indirectly mediated through sympathetic sprouting in the DRG []. Most recently, it is reported that localized inflammation of the DRG up-regulates a number of pro-inflammatory cytokines including IL-6 and induces abnormal sympathetic sprouting in the absence of peripheral nerve injury []. It suggests a possible correlation between inflammatory responses and sympathetic sprouting, which are two well-known mechanisms implicated in various chronic pain states.

In summary, proinflammatory cytokines are involved in the development of inflammatory and neuropathic pain. Just as specific cytokines and their neutralizing antibodies have been introduced into clinical trials for the treatment of stroke, Alzheimer’s disease, autoimmune diseases, wound healing, and amyotrophic lateral sclerosis, one could utilize local or systemic delivery of anti-inflammatory cytokines or inflammatory cytokine antagonists for the treatment of chronic pain. These specific cytokines or antagonists would act to disrupt the hyperexcitability cycle taking place in the sensory neurons, providing a new, non-opioid therapeutic approach for the treatment of pathological pain due to inflammation or peripheral nerve injury.


A Case of Pellagra Associated with Long Term Alcoholism

Content Source: The Journal of Psychiatry and Neurological Sciences

To the Editor,

Pellagra is a systemic, nutritional disease associated with deficiency of vitamin B3 (niacin) and/or tryptophan and often other B vitamins (1). Pellagra is mostly seen in chronic alcoholics as a result of nutritionally poor diet and malabsorption (2). We present a pellagra case with long history of alcohol use, admitted with psychiatric complaints to our clinic.

Mr. A. was a 44 year old, married, primary school graduate male, who was running a coffeehouse. His socioeconomic status was low. His complaints were irritability, nausea, vomiting and loss of appetite. He had been drinking alcohol every day, for 33 years; its amount had increased to about 100cl for the last 15 years. The longest duration of remission was 3 months, when he was 13 years old. He was experiencing sweating, tremor of hands, insomnia, and irritability as withdrawal symptoms. In the last 2 years, periodically, he had problems in focusing and maintaining attention, delay in reaction time in answering any questions. He had depressive symptoms for 1 year and he had attempted suicide. In the last 2 months, he had diarrhea, vomiting, loss of appetite and erythema, followed by dark discoloration on the dorsal surfaces of his hands. On physical examination, hyper-keratotic plaques with well-defined borders on the dorsal surfaces of both hands, squamous lesions between fingers of both feet, loss of villi and hyperemia on the tongue was detected. He had tremor of both hands and wide-based gait. On psychiatric examination, he was confused, his time orientation was disturbed, self care was poor. Affect was restricted; associations and psychomotor activity were slow. The possibility of pellagra was considered as dermatitis, diarrhea and distortion of cognitive functions were observed. Electrocardiography (ECG), complete blood count, routine blood biochemical tests, routine urine tests, thyroid function tests, VDRL, microscopic stool examination, electroencephalography (EEG), vitamin B12 and folate measurements, cranial MRI, echocardiography, esophago-gastro-duodenoscopy were performed and no significant pathology was detected. As the patient’s symptoms did not respond to oral niacin treatment, niacin malabsorption was considered and a mixture of vitamin B1, B2, B6, B12, nicotinamide and dexpanthenol was given by intramuscular injection and a dramatical recovery was observed.

Pellagra is characterized by photosensitive symmetrical skin lesions, gastrointestinal disturbances, neurologic and psychiatric manifestations. The syndrome is known as “4 D’s”: dermatitis, diarrhea, dementia and death (1). Skin lesions seen in pellagra are photosensitive rash, primarily on the dorsal surfaces of the hands, arms, face and feet. In acute phase, skin lesions are erythema and bullae which resemble sunburn (wet pellagra), but after exposure to sun light, progress to chronic, symmetrical, scaled lesions occurs. Typically they are located on the neck (Casal necklace), hands and forearms (pellagra gauntlet) (3). Irritability, concentration problems, anxiety, fatigue, restlessness, apathy and depression are common psychiatric and neurological manifestations. Even uncommon, psychosis can be seen in pellagra, especially in pellagroid encephalopathy mostly encountered in chronic alcoholics. Confusion and eventually death occurs as the disease progresses (4). Gastrointestinal manifestations are fissures on the tongue and mouth, sourness, loss of appetite, dyspepsia and abdominal pain. Enteritis, which can be severe with nausea, vomitting and diarrhea can also be seen (5). Diagnosis is based on patient’s history and physical examination. There are no chemical tests to definitely diagnose pellagra (6).

In conclusion, low socioeconomic status, long duration of alcohol use, poor diet and characteristic findings should suggest pellagra, although it is a rare disease nowadays. It shouldn’t be considered as a disease that is seen only in undeveloped countries and considering pellagra in the differential diagnosis in chronic alcoholics with psychiatric, dermatologic and gastrointestinal symptoms has vital importance.


1. World Health Organization. Pellagra and its prevention and control in major emergencies. Geneva, World Health Organization, 2000 (document WHO/NHD/00.10).

2. Stratigos JD, Katsambas A. Pellagra: a still existing disease. Br J Dermatol 1977; 96:99-106.

3. Pipili C, Cholongitas E, Ioannidou D. The diagnostic importance of photosensivity dermatoses in chronic alcoholism: Report of two cases. Dermatol Online J 2008; 14:15.

4. Cook CC, Hallwood PM, Thomson AD. B Vitamin deficiency and neuropsychiatric syndromes in alcohol misuse. Alcohol Alcohol 1998; 33:317-336.

5. Karthikeyan K, Thappa DM. Pellagra and skin. Int J Dermatol 2002; 41:476-481.

6. Hegyi J, Schwartz RA, Hegyi V. Pellagra: Dermatitis, dementia, and diarrhea. Int J Dermatol 2004; 43:1-5.

Nutrient Deficiency Diseases

~Content Source

Deficiency diseases – Scurvy, beriberi, pellagra, rickets, goiter, protein (amino acid) deficiencies, marasmus and kwashiorkor.

Nutrient deficiency diseases occur when there is an absence of nutrients which are essential for growth and health. Lack of food leading to either malnutrition or starvation gives rise to these diseases. Another cause for a deficiency disease may be due to a structural or biological imbalance in the individual’s metabolic system.

There are more than 50 known nutrients in food. Nutrients enable body tissues to grow and maintain themselves. They contribute to the energy requirements of the individual organism and they regulate the processes of the body. Carbohydrates, fats, and proteins provide the body with energy. The energy producing component of food is measured in calories. Aside from the water and fiber content of food, which are also important for their role in nutrition, the nutrients that serve functions other than energy production can be classified into four different groups: vitamins, fats, proteins, and minerals. All are necessary for proper body function and survival.

Early vitamin deficiency diseases

Polish-born Casimir Funk (1884-1967) originated the word vitamin in 1912, spelling it as vitamine, because he thought they were part of a group of organic compounds containing nitrogen, called amines. The final –e was later dropped in 1920 at the suggestion of the English nutritionist Jack Cecil Drummond who pointed out that these trace-like substances found only in food and essential for good health were not always amines. By 1914 Funk theorized that beriberi, scurvy, and pellagra were caused by a vitamin deficiency.


Scurvy is one of the oldest vitamin deficiency diseases recorded and the first one to be cured by adding a vitamin to the diet. Scurvy was a common malady of sailors of the age of exploration of the New World. It has been recorded that Vasco da Gama was supposed to have lost half of his crew to scurvy in his journey around the Cape of Good Hope at the end of the fifteenth century and Richard Hawkins reported that he lost 10,000 sailors from the disease a century later.

The main symptom of scurvy is hemorrhaging. Hemorrhage marks appear as spots under the skin or bruises, given the medical terms of petechiae and ecchymoses. The gums are swollen and usually become infected (gingivitis). Bleeding can take place in the membranes covering the large bones as well as in the membranes of the heart and brain. Wounds heal slowly and the bleeding in or around vital organs can be fatal. The disease is slow to develop and is manifested early by fatigue, irritability, and depression.

In 1747 a British naval physician, James Lind, in a response to a an outbreak of scurvy conducted a controlled experiment. He took 12 of the sailors who had developed scurvy and divided them up into six groups and gave each pair different medicines such as nutmeg, cider, seawater, and vinegar, while others were given lemons or oranges. The two men given the oranges and lemons both completely recovered in about a week after the experiment.

His Treatise of the Scurvy published in 1753 is the first example of a controlled clinical trial experiment. In his treatise, Lind gave a thorough review of other authors who had written on scurvy along with a careful clinical description of the condition. It was not until the end of the eighteenth century that the British navy finally had its sailors drink a daily portion of lime or lemon juice to prevent scurvy.

Vitamin C (ascorbic acid) is necessary for collagen formation, which is the protein component of connective tissue, strong blood vessels, healthy skin and gums, formation of red blood cells, wound healing, and the absorption of iron. In addition to scurvy, other scurvy-like conditions can develop from a deficiency of vitamin C, such as adult acne, easy bruising, sore gums, and hemorrhages around bones. Good sources for vitamin C are citrus fruit, broccoli, strawberries, cantaloupe, and other fruits and vegetables.


Discovering the causes for beriberi became part of the history of discovering vitamins. Christian Eijkman (1858-1930) was a Dutch physician who was a member of a government commission sent to the East Indies in the 1880s to study the disease beriberi, which was prevalent in southeast Asia, where the main diet is comprised of unenriched rice and wheat.

There are three forms of this disease: infantile beriberi, wet beriberi, and dry beriberi. Infantile beriberi occurs when a mother who breast feeds her child is lacking vitamin B1 thiamine. The mother who nurses the child may not manifest the disease, but the deficiency occurs through the breast feeding and the child usually dies after the fifth month. In the childhood and adult versions of the disease there is a preliminary condition of fatigue, loss of appetite, and a numb tingling feeling in the legs. This condition can then lead to either wet or dry beriberi.

In wet beriberi there is an accumulation of fluid throughout the body and a rapid heart rate that can lead to sudden death. In dry beriberi there is no fluid swelling, but there is a loss of sensation and a weakness in the legs. The patient first needs to walk with the aid of a stick and then becomes bedridden and easy prey to an infectious disease.

In Eijkman’s laboratory he noticed that some of the fowl he was experimenting with developed paralysis and polyneuritis, as in the dry form of beriberi. The director of the hospital forbade Eijkman from feeding these birds with table scraps which consisted mainly of polished rice. He therefore began to feed them with whole rice, after which he noticed that they regained their movement and there was no recurrence of paralysis.

The idea that the birds had some form of beriberi was rejected by Eijkman’s colleagues. His explanation for the cure was that the polished rice had some toxin in it which the unpolished rice did not have. This explanation was rejected by a fellow researcher, Gerrit Grijns (1865-1944), who also stayed on to study the disease after the commission had already left. He found that when the chickens were taken off the rice diet completely and feed with meat instead, they did not develop the characteristic paralysis, but if the meat were overcooked, then the condition would reappear. In 1901 Grijns showed that beriberi could be cured by putting the rice polishings back into the rice.

Vitamin B1 (thiamine) prevents the disease or symptoms of beriberi. Food sources for this vitamin are meats, wheat germ, whole grain and enriched bread, legumes, peanuts, peanut butter, and nuts.


Pellagra is a vitamin deficiency disease associated with poverty. The symptoms of pellagra are referred to as the “three D’s”: diarrhea, dermatitis, and dementia. If disease is not treated it may lead to death. Gaspar Casal (c. 1691-1759) was the first to publish a thorough explanation of pellagra in 1762 after his death. He studied and wrote about the disease which he observed in a region of Spain where it was called “mal de la rosa,” because of the reddened dermatitis which appeared around the back of the neck. Even though the belief of his time was the disease was caused by an infection, Casal believed origins were from inadequate nutrition.

The popular belief that pellagra was caused by infection lasted from the sixteenth century to the early twentieth century until Joseph Goldberger (1881-1929) a member of the United States Public Health Service studied the high numbers of cases in the southern United States. Goldberger established that pellagra was caused by an insufficient amount of niacin (vitamin B3) also known as nicotinic acid and the active form of niacin that the body uses called niacinamide.


Rickets is a bone disease deficiency caused by a lack of vitamin D, called the “sunshine” vitamin because it is the only vitamin that can be produced by the effects of sunlight on the skin. It was a common disease of infants and children, but since all milk and infant formulas have vitamin D added to them, it is rarely seen today. In rickets, legs will become bowed by the weight of the body and the wrists and ankles are thickened. The teeth are badly affected and take a longer time to come in. All the bones are affected by not having sufficient calcium and phosphorous for their growth and development. Lack of exposure to sunlight, which helps to produce vitamin D, is a major cause for childhood rickets. Crowded slum conditions in areas where there was little or no sunlight were responsible for its appearance in the earlier stages of the industrial revolution.

An adult version of rickets caused by a deficiency of vitamin D, calcium, and phosphorous is called osteomalacia. The bones become soft and deformed and there is rheumatic pain. The disease is observed in the Middle East and Asia more so than in western countries. The way to prevent rickets and other bone diseases such as osteoporosis is a combination of calcium, phosphorous, and vitamin D.

Other vitamin deficiency diseases

Night blindness or the difficulty of seeing in dim light is caused by a deficiency in vitamin A which helps in the formation of visual purple needed by the eyes for night vision. The deficiency can also cause glare blindness when the eye is either exposed to too much light or a sudden change in the amount of light when entering a darkened room. Another eye disease caused by vitamin A deficiency is xerophthalmia which can lead to blindness. This condition affects the cells of the cornea, other eye tissues, and the tear ducts, which stop secreting.

Vitamin A deficiency can create a number of adverse skin conditions, problems with tasting and smelling, and it may also cause difficulties with the reproductive system.

Vitamin E and K deficiencies are rare. Vitamin E protects against substances that oxidize quickly and vitamin K promotes normal blood clotting. Vitamin B12 (cobalamin) provides protection against pernicious anemia and mental disturbances. Vitamin B 6can also protect against anemia as well as dermatitis, irritability, and convulsions.

Mineral deficiency diseases

There are about 25 mineral elements in the body usually appearing in the form of simple salts. Those which appear in large amounts are called macro minerals while those that are in small or trace amounts are micro minerals. Some that are essential are calcium, phosphorous, cobalt, copper, fluorine, iodine, iron, sodium, chromium, and tin. Aluminum, lead, and mercury are not as essential.


Iodine is necessary for the proper functioning of the thyroid gland which controls the body’s basal metabolism rate through its production of two hormones, thyroxine, and triiodinethyronine. Without a sufficient amount of iodine in the diet the gland begins to enlarge its cells in its efforts to produce the hormone, thus producing a goiter, which is a swelling around the neck. Certain regions lack iodine in the soil which leads to cretinism, the physical and mental development of an infant passed on from the lack of iodine in the mother’s diet.

Protein (amino acid) deficiencies

Proteins are needed in the body for amino acids. Proteins are broken down in the digestive system to form amino acids which are then absorbed by the rest of the body to form new proteins in the form of vital body tissues such as muscle, connective tissue, and skin. There are two types of protein, fibrous and globular proteins. Fibrous protein is insoluble and goes into making the structural tissues of the body. Globular protein forms amino acids that become enzymes and hormones and other vital parts of cellular functioning within the body.

Adults rarely suffer from protein deficiency diseases unless there is an impairment in the intestinal tract, but in countries plagued by insufficient food children will develop protein deficiency diseases that lead to very high mortality rates.

Marasmus and kwashiorkor

A specific wasting away disease caused by protein deficiency in third world countries that lack adequate food supplies is called kwashiorkor. It is a word which describes the condition of an infant who has to be weaned away after a year to make room for the next baby. The weaning food, which is mainly sugar and water or a starchy gruel lacks protein or has a poor quality of protein. The weaning diet for these young children leads to other nutrient deficiency diseases as well.

Symptoms of kwashiorkor are apathy, muscular wasting, and edema. Both the hair and the skin lose their pigmentation. The skin becomes scaly and there is diarrhea and anemia, and permanent blindness can result from this condition. Marasmus is another condition of a wasting away of the body tissues from the lack of calories as well as protein in the diet. In marasmus the child is fretful rather than apathetic and is skinny rather than swollen with edema. Aside from contrasting symptoms between the two diseases, there may be converging symptoms which would be described as marasmic kwashiorkor.

There is a wide variation of deficiencies between energy and protein deficient diseases as in the cases described by marasmus and kwashiorkor. The term protein-energy malnutrition (PEM) is used to describe those differences. PEM is the result of poverty as well inadequate information on diet. In some countries there is the mistaken belief that the child should not be given high protein food, which is served to the father, while the child drinks the fluid the meat was cooked in.

In cases of severe PEM it is necessary to hospitalize the child and to administer antibiotics to prevent infections which accompany the condition. Diets rich in protein should be continued after hospitalization, using skimmed milk powder for an energy basis. Legumes (beans) and fish meal are also good sources for protein. Social and political problems have to be managed to allow relief workers to help and to provide an ongoing source of food preparations that can be consumed for adequate nourishment by those in need.

Treatment and prevention

The amounts of most nutrients, especially vitamins, needed to both prevent and treat deficiency diseases are small. The average intake of 1mg of vitamin B1 is sufficient to prevent a deficiency disease of that vitamin, while 10mg of B1 could cure an advanced case of someone about to die of beriberi. Although small doses of vitamins cure deficiencies, large doses of some vitamins such as A and D can be harmful since these two vitamins are already stored by the liver. Vitamins A and D are fat soluble vitamins and can accumulate to the point of becoming toxic. Most other vitamins are water soluble and are excreted in the urine throughout the day.

Diet and supplements

Most nutritionists insist on a well-balanced diet consisting of the major food substances as an effective and economical way of obtaining nutrients for health. On the other hand, advocates of health food stores maintain that the FDA’s required daily allowances (RDAs) for nutrients are much too low and that cultivation of much of our food supply and its preparation robs our diet of much of its nutrient value.

The American Dietetic Association (ADA) recommends that nutrient needs should come from a variety of foods taken from different dietary sources rather than self-prescribed vitamin supplementation. In order to avoid either the problem of nutrient deficiencies or excesses they recommend that physician’s or licensed dietician’s should be the source of prescribing supplementation.

The ADA, however, does make allowances for supplement usage under the following conditions: Iron supplements may be required by women when there is excessive menstrual bleeding. Pregnant and breast-feeding women need supplements, especially iron, folic acid, and calcium. People who are dieting and are therefore are on very low calorie diets may require supplementation if they are not getting the right amount of the nutrients they need. Vegetarians may need boosts of vitamin B-12, calcium, iron, and zinc. Newborns are sometimes given vitamin K to prevent abnormal bleeding. Those people who have diagonsised disorders or diseases or are being treated with medications which affects the absorption or metabolism of the nutrient may require supplementation.


Amino acid —An organic compound whose molecules contain both an amino group (-NH2) and a carboxyl group (-COOH). One of the building blocks of a protein.

Calcium —An essential macro mineral necessary for bone formation and other metabolic functions.

Controlled experiment —Also called a controlled trial. The dividing into groups of experimental subjects to see what the effects of a drug will be when tested along with a dummy drug or placebo (a drug other than the one being tested).

Dermatitis —An inflammation of the skin. A symptom of vitamin deficiency.

Edema —An abnormal collection of fluids in the body tissues. One of the forms of the disease beriberi called wet beriberi.

Essential nutrients —Those nutrients that must be obtained from food for good health and to prevent nutrient deficiency diseases.

Iodine —A mineral necessary for the proper functioning of the thyroid gland.

Niacin —An essential B vitamin needed to prevent pellagra.

Night blindness —Inability to see at night due to a vitamin A deficiency.

Recent research on vitamins A and C

Research using 22,000 physicians under the supervision of the Department of Medicine at Harvard is studying the long-term effects of beta carotene (vitamin A) in lowering the incidence of cancer and boosting resistance to infection. It is also being studied in the treatment of AIDS. Beta carotene is a safer version of vitamin A than the preformed oil form called retinol. It is found in carrots, sweet potatoes, broccoli, spinach, collards, turnip greens, kale, and many other vegetables that.

Vitamin C, also known as ascorbic acid, is used as a supplement by more people than any other supplement. Its popularity is due to the work of the two-time Nobel laureate, Linus Pauling who maintained that vitamin C was effective in preventing and lessening the effect of colds and in the treatment of cancer. Pauling’s vitamin C program called for megadoses that far exceeded the government’s RDA recommendations. Pauling recommended a daily dose of between 2,000 and 9,000 milligrams (mg). The National Research Council recommends 60 mg for adult daily and 100 mg for smokers.

The discovery of micro nutrition was made in the early twentieth century as a result of finding the cure for certain diseases, the nutrient deficiency diseases such as scurvy, beriberi, and pellagra. The new dimensions of fully understanding and using our knowledge of nutrients remain to be established from the ongoing research in this area of nutritional science.



Encyclopedia of Human Nutrition, edited by Benjamin Caballero, et al. London: Academic Press, 2005.

Hendler, Sheldon S. The Doctor’s Vitamin and Mineral Encyclopedia. New York: Simon and Schuster, 1990.

Kok, Frans J., et al. Introduction to Human Nutrition. Oxford: Blackwell Publishing, 2002.

Williams, Sue R. Nutrition and Diet Therapy. Boston: Mosby College Publishing, 1989.

Jordan P. Richman

Production of Melanin Pigment by Fungi and Its Biotechnological Applications

This is fascinating. To me anyways. It goes a long way in explaining why each and every one of us has differences in skin pigmentation. We really are all just the same. It’s our microbes that are different.

By Sandra R. Pombeiro-Sponchiado, Gabriela S. Sousa, Jazmina C. R. Andrade, Helen F. Lisboa and Rita C. R. Gonçalves

Submitted: June 1st 2016Reviewed: December 28th 2016Published: March 1st 2017

DOI: 10.5772/67375


Production of the microbial pigments is one of the emerging fields of research due to a growing interest of the industry for safer products, easily degradable and eco-friendly. Fungi constitute a valuable source of pigments because they are capable of producing high yields of the substance in the cheap culture medium, making the bioprocess economically viable on the industrial scale. Some fungal species produce a dark-brown pigment, known as melanin, by oxidative polymerization of phenolic compounds, such as glutaminyl-3,4-dihydroxybenzene (GDHB) or catechol or 1,8-dihydroxynaphthalene (DHN) or 3,4-dihydroxyphenylalanine (DOPA). This pigment has been reported to act as “fungal armor” due to its ability to protect fungi from adverse conditions, neutralizing oxidants generated in response to stress. Apart from the scavenging activity, melanin exhibits other biological activities, including thermoregulatory, radio- and photoprotective, antimicrobial, antiviral, cytotoxic, anti-inflammatory, and immunomodulatory. Studies have shown that the media composition and cultivation conditions affect the pigment production in fungi and the manipulation of these parameters can result in an increase in pigment yield for large-scale pigment production. This chapter presents a comprehensive discussion of the research on fungal melanin, including the recently discovered biological activities and the potential use of this pigment for various biotechnological applications in the fields of biomedicine, dermocosmetics, materials science, and nanotechnology.


  • fungi
  • pigment
  • melanin
  • biological activity
  • industrial applications

1. Introduction

Considering the harmful effects of synthetic dyes on human health and to the environment, developmental process for obtaining pigments from natural sources has become significant worldwide. Microbial pigments have gained attention owing to a growing interest of the industry in safer products, easily degradable, eco-friendly and do not cause harmful effects. The pigment production from microorganisms is considered more advantageous because it is a more efficient and cost-effective process than chemical synthesis of pigments. Microorganisms are also more feasible sources of pigments in comparison to pigments extracted from plants and animals because they do not have seasonal constraints, do not compete for limited farming land with actual foods, and can be produced easily in the cheap culture medium with high yields [16]. Besides, the microorganisms produce an extraordinary range of pigments that include several chemical classes such as carotenoids, melanins, flavins, phenazines, quinones, monascins, violacein, or indigo, as shown in Table 1.

Pigment Microorganism
Indigoidine (blue-green) Streptomyces aureofaciens CCM 323, Corynebacterium insidiosum
Carotenoid (orange) Gemmatimonas aurantiaca T-27
Melanin (black-brown) Kluyveromyces marxianusStreptomyces chibanensisCryptococcus neoformansAspergillus sp., Wangiella dermatitidisSporothrix schenckii, and Burkholderia cepacia
Prodigiosin (red) Serratia marcescensRugamonas rubraStreptoverticillium rsubrireticuliSerratia rubidaeaVibrio psychroerythrusAlteromonas rubra, and Vibrio gazogenes
Zeaxanthin (yellow) Staphylococcus aureusVibrio psychroerythrusStreptomyces sp., and Hahella chejuensis
Canthaxanthin (orange) Monascus roseusBradyrhizobium sp.
Xanthomonadin (yellow) Xanthomonas oryzae
Astaxanthin (red) Phaffia rhodozymaHaematococcus pluvialis
Violacein (purple) Janthinobacterium lividum
Anthraquinone (red) Paecilomyces farinosus
Halorhodopsin and rhodopsin (pink Halobacterium halobium
Rosy pink Lamprocystis roseopersicina
Violet/purple Thiocystis violaceaThiodictyon elegans
Rosy peach Thiocapsa roseopersicina
Orange brown Allochromatium vinosum
Pink/purple violet Allochromatium warmingii

Table 1.

Pigments produced by different microorganisms. Adapted from Ref. [3].

Among microbial species, fungi represent an economically significant source of these compounds because they can act as microbial cell factories producing high yields of metabolites with great diverse chemical structures combined with ease of large-scale cultivation [79].

As shown in Table 1, some fungal species produce a dark-brown pigment, known as melanin. In general, this pigment is located in the outermost layer of the cell wall associated with chitin (referred as cell wall-bound melanin), but in some fungi, melanin can also be found outside the fungal cell, usually in the form of granules in culture fluids [10].

Fungal melanins are negatively charged, hydrophobic pigments of high molecular weight formed by oxidative polymerization from phenolic and/or indolic compounds, such as glutaminyl-3,4-dihydroxybenzene (GDHB) or catechol or 1,8-dihydroxynaphthalene (DHN) or 3,4-dihydroxyphenylalanine (DOPA). Most Ascomycota fungi synthesize DHN-melanin from the polyketide synthase pathway, whereas few species are able to produce melanin through L-DOPA, in a pathway that resembles mammalian melanin biosynthesis [1113].

The melanin pigment is not essential for fungal development, but it has been reported to act as “fungal armor” due to its ability to protect the microorganisms from harmful environmental conditions. In vitro studies have shown that melanized fungi are more resistant to UV light-induced and oxidant-mediated damages, temperature extremes, hydrolytic enzymes, heavy metal toxicity, and antimicrobial drugs than those nonmelanized [101417]. Recent studies have shown that in industrial and roadside areas, there is an increase in the proportion of dark melanin-containing fungi, as Cladosporium and Alternaria, which were more resistant to contamination by heavy metals and unsaturated hydrocarbons. Radionuclide contamination also led to a change in fungal communities, with an increased proportion of melanized fungi. For example, melanized fungal species as Cladosporium spp., Alternaria alternataAureobasidium pullulans, and Hormoconis resinae were found to colonize the walls of the damaged reactor at Chernobyl where they are exposed to a high constant radiation field [1819].

The presence of melanin in the cell wall is also correlated with enhanced virulence of parasitic fungi, as Paracoccidioides brasiliensisSporothrix schenckii, and Exophiala (Wangielladermatitidis [172021]. This pigment protects the conidia against digestion by proteases and hydrolases secreted by competitive microorganisms or against bactericidal and fungicidal proteins of animal origin, such as defensins, magainins, or protegrins [22]. This effect was observed for Cryptococcus neoformans, whose in vitro melanization has been associated with resistance against host effector cells, oxidants, microbicidal peptides, and amphotericin B [2325], and in Wangiella (Exophialadermatitidis, when the polyketide synthase gene WdPKS1 associated on melanin production was disrupted, this strain has become more susceptible to voriconazole and amphotericin B [26]. Others studies suggest that melanin contributes to fungal pathogenesis because this pigment alters the host defense response mechanisms, decreases phagocytosis, and reduces the toxicity of microbicidal peptides, reactive oxygen species, and antifungal drugs as well as to play a significant role in fungal cell wall mechanical strength [2728].

Although the molecular structure of fungal melanin remains enigmatic, significant progress has been made in understanding particular aspects of its macro- and microstructure. These advances allow to elucidate the molecular mechanisms of the various biological functions of melanin [22]. Studies have shown that the effect of melanin enhancing the survival of fungi under adverse conditions can be mainly due to its powerful free radical scavenger properties, acting as a “sponge” for other free radicals generated by the fungus in response to environmental stress [202930]. Apart from this scavenging ability, melanin exhibits other biological activities, including thermoregulatory, photoprotective, antimicrobial, antiviral, cytotoxic, anti-inflammatory, radioprotective, and immunomodulatory [1317183134].

Since melanin has characteristics of functional materials and bioorganic, a growing number of researchers see this pigment with great interest, taking advantage of their properties for numerous biotechnological applications in cosmetics, pharmaceutical, electronic, and food processing industries [121935].

The purpose of this chapter involves a comprehensive discussion of the research on fungal melanin, including the recently discovered biological activities and the potential use of this pigment for several biotechnological applications. Additionally, we discussed the ways to explore the metabolic potential of the pigment-producing fungi by manipulation of cultivation conditions to improve performance of the process, increasing yields, and reducing cost, for large-scale production.

2. Factors influencing the melanin production

Microbial pigment production is now one of the emerging fields of research due to its potential for various industrial applications, as foodstuff, cosmetics, pharmaceutical, and textile manufacturing processes. However, it is known that for the success of microbial fermentation processes, it is necessary to choose the correct productive culture strain and to determine the appropriate cultivation conditions [4836].

An ideal pigment-producing microorganism should be capable of using a wide range of C and N sources; must be tolerant to pH, temperature, and minerals concentration; and must give reasonable pigments yield. The nontoxic and nonpathogenic natures, coupled with easy separation from cell biomass, are also preferred qualities. The potential of using filamentous fungi as pigment sources is due to their extraordinary metabolic versatility because they can be cultivated over a wide range of temperatures (10–50°C), pH (2–11), salinity (0–34%), and water activity (0.6–1) and under oligotrophic or nutrient-rich conditions. They can grow in different culture systems (submerged and solid), and fermentation protocols have been established for large-scale industrial processes. In addition, these organisms can be genetically modified to increase productivity and quality of the produced pigments [3738].

In order to improve performance and reduce the cost of pigments produced by microbial fermentation, it is essential to identify the nutritional and physical factors that have a greater influence on the cell growth and metabolite biosynthesis [463940].

Several studies have shown that the composition of the growth medium, nature and concentration of carbon and nitrogen sources, minerals, vitamins, temperature, pH, the presence of oxygen and aeration, light, stress, and irradiation, among others, affect the growth and pigment production in fungi and that the manipulation of the culture conditions can result in enhanced pigment production [4147].

Experimental evidences indicate that the growth temperature influences the performance of the pigment production process, but this effect depends on the type of organism. Pseudomonas requires 35–36°C for its growth and pigment production, while in Monascus purpureus, maximum pigment production was observed at 30°C with a reduction of the yield at 37°C [48]. Another study in Monascus sp. J101 reported that the yield of pigment at 25°C was ten times higher than at 30°C, probably due to long growing (120 hours) and lower viscosity of the broth at 25°C compared to 30°C [49]. Studies developed in our laboratory, using a melanin-overproducing mutant (MEL1) from Aspergillus nidulans fungus, showed that the higher production of pigment occurred at incubation temperature of 28°C compared to 37°C [50].

Researches support that the pH of the medium also affects the growth of fungi and type of pigment produced. In species of Monascus, the pH influences the yield and quality of the produced pigment, with the highest red pigment excretion and production at alkaline pH [5152]. Studies on wood-inhabiting fungi indicate that pH of the substrate potentially plays an important role in fungal melanin formation. Fungi Trametes versicolor and Xylaria polymorpha tested on wood substrates produced maximum pigmentation at the pH range 4.5–5.0, except for Scytalidium cuboideum, which produce maximum intensity of red pigment at pH 6 and blue pigment at pH 8 [53]. In our study with the hypermelanized mutant (MEL1) from Anidulans, we observed an increase in the production of pigment when the initial pH of the culture was at 6.8 compared to pH 8.0 [50]. Metabolically, the effects of pH and temperature on fungal pigment production is associated with changes in protein activity, so that the culture conditions may control certain activities such as cell growth, production of primary and secondary metabolites, fermentation, and oxidation processes of the cell [54].

The influence of light on intra- and extracellular pigment production was studied in five pigment-producing fungi: M. purpureusIsaria farinosaEmericella nidulansFusarium verticillioides, and Penicillium purpurogenum [55]. These authors concluded that the cultivation in the total absence of light increased biomass and production of extracellular and intracellular pigments in all fungi. The fungi grown under red light have no effect, and green or yellow light resulted in worsening effect in all the fungi, thus postulating the existence of photoreceptors responsive to dark and light in all the fungi. In a similar study, [56] noted that the production of pigment by Monascus species also was favored when the fungus was grown in the dark.

Some studies report that the pigment synthesis requires proper aeration probably related to the oxygen dependency of some enzymatic reactions responsible for the production of pigment. In Monascus ruber, it was observed that the highest levels of pigments production were obtained at an aeration rate of 0.05 L min−1, which appeared to be clearly sufficient for providing the fungus with oxygen and removing carbon dioxide [57]. In our studies, it was noted that no melanin pigment production takes place during stationary cultivation of hypermelanized mutant (MEL1) from Anidulans, indicating that the formation of this pigment involves the oxidative polymerization of the precursors [50].

Carbon and nitrogen are necessary for cellular metabolism, and these sources are related to the formation of biomass, the type produced pigment, and the yield of the desired substance. These nutrients may regulate the expression of genes of interest and activate important metabolic pathways for the production of pigments [455859]. In general, glucose, an excellent carbon source for growth, interferes with the formation of many secondary metabolites, including pigments. For example, the pigment production by Penicillium sp. was evaluated in the presence of 10 different carbon sources, and the maximum mycelial growth was obtained with fructose, whereas the maximum pigment production was obtained with soluble starch [60]. This result shows that the increased biomass does not necessarily result in increased pigment production because pigments produced by fungi are secondary metabolites whose production usually occurs at the late growth phase (idiophase) of these microorganisms [61]. The pigment production capability of fungal species belonging to the genera PenicilliumAspergillusEpicoccumLecanicillium, and Fusarium was evaluated in different culture media, and the results showed that the complex media, as potato dextrose (PD) and malt extract (ME), favored increased pigment production [47]. According to the authors, these media contain nutrients that can regulate the expression of genes of interest and activate metabolic pathways important for the production of pigments.

Studies have demonstrated that the promoting or repressing effect of a nitrogen source on pigment production is strain dependent. It has been reported that various types of peptone, used as a nitrogen source, are able to promote an increase in the production of pigments in many species of fungi [55596263]. However, M. purpureus was not able to grow in media containing peptone, and a maximum yield of the pigment was achieved when the media were supplemented with yeast extract (1%) and monosodium glutamate (5%) as nitrogen source [41]. In M. ruber, the use of glutamic acid as a nitrogen source showed promising results, either as stimulating the accumulation of extracellular pigments or contributing to increase the efficiency of the pigment production process [45]. The production of high amounts of extracellular melanin by the fungus Gliocephalotrichum simplex was obtained in cultures supplemented with tyrosine (2.5%) and peptone (1%) [64].

The optimization of medium composition is an important strategy to increase pigment production because some sources of carbon and nitrogen can be more easily assimilated and promote higher yields of the desired product. During the optimization experiments to enhance the production of melanin by Auricularia auricula, it was observed that soluble starch, tyrosine, peptone, CaCO3, and K2HPO4 had positive effects, while glucose, (NH4)2SO4, MgSO4, CuSO4, and FeSO4 negatively impacted melanin production [46]. In other study with A. auricula, it was observed that yeast extract, tyrosine, and lactose have significant effects on pigment production and the optimization of medium resulted in 2.14-fold higher melanin concentration than that of the unoptimized medium [65].

Since the substrates for the production of pigment strongly influence the cost of the bioprocess, there is a need to select cheap and efficient substrates to make the process economically viable on the industrial scale. Large amounts of agro-industrial residues generated from diverse economic activities have attracted strong industry interest on the utilization of these residues as inexpensive substrates to support the growth of microorganisms in bioprocesses. This strategy may represent an added value to the industry and also helps in solving pollution problems, reducing or preventing their disposal in the environment [16667].

Various studies have reported the successful utilization of agro-industrial residues for the production of fungal pigments. The use of corn cob powder as a substrate for production of pigments by M. purpureus resulted in greater pigment production [68] than other substrates, as jackfruit seed [69], corn steep liquor [70], and grape waste [71]. In the black yeast Hortaea werneckii, it was observed that rice bran acts as the cheapest source for increased production of melanin by than wheat bran and coconut cake [72]. Wheat bran extract, L-tyrosine, and CuSO4 represent the best combination of medium components to obtain the maximum melanin yield from the fungus A. auricula in submerged culture [73]. A study conducted in our laboratory evaluated the use of corn steep liquor, sugarcane bagasse, and molasses as nutritional source on pigment production by melanin-overproducing mutant (MEL1) from Anidulans. We observed that, in the presence of 0.2% corn steep liquor, an increase in the pigment production occurred, while a high yield of biomass was obtained at a concentration of 2%. The supplementation of medium with molasses and sugar cane bagasse hydrolysate did not have a positive effect on pigment production but promoted an increase in the fungal growth. These results indicate that corn steep liquor contains substances that stimulate the synthesis of pigment and it represents a low-cost fermentation medium for large-scale production of the pigment melanin by MEL1 mutant for future industrial applications [74].

3. Pathways of melanin biosynthesis

Various techniques, including electron paramagnetic resonance [75], X-ray diffraction [76], infrared, ultraviolet and visible spectroscopy [77], and nuclear magnetic resonance [78], have been used to elucidate the melanin structure from different organisms. These studies have shown that fungi can produce different types of melanins by oxidative polymerization of phenolic or indolic compounds [1127].

Melanin in cell walls of Basidiomycotina is derived from phenolic precursors, as glutaminyl-3,4-dihydroxybenzene (GDHB) or catechol. In the parasitic fungus Ustilago maydis, polymerization of catechol dimers with the formation of fibrils of melanin was shown [79]. The precursor of melanin in Agaricus bisporus and other Basidiomycetes is a metabolite of the shikimic acid pathway-γ-glutaminyl-4-hydroxybenzene oxidized under the action of peroxidase and/or phenolase into γ-glutaminyl-3,4-benzoquinone, followed by its polymerization [80]. C. neoformans, a pathogenic basidiomycetous yeast, is known to synthesize DOPA-melanin when o-diphenolic compounds, such as 3,4-dihydroxyphenylalanine, are present in the culture medium. This fungus may use a wide array of substrates, such as D- and L-dopamine [81], homogentisic acid [82], catecholamines, and other phenolic compounds [83], maximizing its ability to produce melanin. Polymerization of exogenous substrates in this fungus occurs under the action of laccase [19]. However, it is important to emphasize that different properties are observed for melanins derived from different substrates. Comparison of the catecholamines L-dopa, methyldopa, epinephrine, and norepinephrine shows differences in term of color, yield, and thickness of the cell wall melanin layer. It was also observed that the pigments vary in the strength of the stable free radical signal detectable by EPR [1383].

In the Ascomycota fungi, melanin pigment is generally synthesized from the pentaketide pathway in which 1,8-dihydroxynaphthalene (DHN) is the immediate precursor of the polymer, as described by Bell and Wheeler [11] based on genetic and biochemical evidence obtained from Verticillium dahliae and W. dermatitidis [8485]. Figure 1 shows a general model for fungal dihydroxynaphthalene (DHN)-melanin biosynthesis. In this pathway, the polyketide synthase (PKS) converts malonyl-CoA to 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN), which undergoes several reduction and dehydration reactions to produce scytalone, 1,3,8-trihydroxynaphthalene (THN), and vermelone. A further dehydration step leads to the intermediate 1,8-dihydroxynaphthalene (DHN), which is polymerized to DHN-melanin, possibly by a laccase enzyme [101327].

Figure 1.

The biosynthetic pathway of fungal dihydroxynaphthalene(DHN)-melanin. Scheme adapted from Ref. [13].

However, some species of this class, including Cladosporium resinaeEpicoccum nigrumHendersonula toruloideaEurotium echinulatumHumicola grisea, and Hypoxylon archeri, do not produce this type of pigment [11288688]. In the genus Aspergillus, DHN-melanin has not been identified in some members, as Anidulans and Aniger. Bull [89] identified dopachrome (indole 5,6-quinone 2-carboxylic acid) and melanochrome (indole 5,6-quinone), which are intermediates in the DOPA-melanin pathway, in Anidulans mutants defective in the production of melanin. Other studies confirmed the indolic nature of the melanin produced by Anidulans [1190]. In Anidulans strains, one tyrosinase was identified as the enzyme responsible for the production of melanin pigment, based on its substrate specificity (DOPA substrate) and susceptibility to inhibitors [9192]. In a recent study, our group characterized the pigment produced by Anidulans mutants as DOPA-melanin according to the results obtained with specific inhibitors of DHN- and DOPA-melanin pathways [93].

The production of DOPA-melanin has also been investigated in other fungi such as Neurospora crassa [94], Podospora anserina [95], Anidulans [91], Aoryzae, and Cneoformans [96]. A biosynthesis pathway for fungal DOPA-melanin, proposed by [11], is shown in Figure 2, which strongly resembles the pathway found in mammalian cells, though some of the details may differ.

Figure 2.

The biosynthetic pathway of the dihydroxyphenylalanine (DOPA)-melanin in fungi. Scheme adapted from Ref. [13].

In this pathway, there are two possible starting molecules, L-dopa and tyrosine. If L-dopa is the precursor molecule, it is oxidized to dopaquinone by laccase. If tyrosine is the precursor, it is first converted to L-dopa and then dopaquinone. The same enzyme, tyrosinase, carries out both steps. Dopaquinone, a highly reactive intermediate, forms leucodopachrome, which is then oxidized to dopachrome. Hydroxylation (and decarboxylation) yields dihydroxyindoles, which can polymerize spontaneously to form DOPA-melanin [102797].

Some fungi have more than one biosynthetic pathway of melanins. For example, Aspergillus fumigatus synthesizes DHN-melanin [98] and also produces a second type of melanin, piomelanins, from homogentisic acid by the tyrosine degradation pathway that protects the cell wall of hyphae from ROS, and gray-green DHN-melanins determine the structural integrity of the cell wall of conidia and their adhesive properties [99]. In Agaricus bisporus, melanins are formed from DOPA by tyrosinase and from γ-glutaminyl-4-hydroxybenzene by peroxidase and phenolase [100].

The extracellular fungal melanin, which is found in culture fluids usually in the form of granules, can be formed from some culture components, which are autoxidized or are oxidized by phenoloxidases released from the fungus during autolysis [101127].

4. Biological activities of melanin

Despite the difference in their origins, melanin pigments have a number of common characteristics that allow them to fulfill their protective function. Several biological functions of melanins are closely associated to their chemical composition and structure. The presence of unpaired electrons in the melanin structure is responsible for various properties, including antioxidant, semiconductor, optical, electronic, and radio- and photoprotective [19].

The effect of melanin enhancing the survival of fungi under adverse conditions is mainly due to its function as an extracellular redox buffer, which can neutralize oxidants generated by the fungus in response to environmental stress [19]. It has been reported that melanin contributes for virulence of C. neoformans, protecting the pathogen against free radicals generated immunologically [29]. In W. dermatitidis and A. alternata, melanin confers resistance to oxidants permanganate and hypochlorite, representing a key role in pathogenesis of infections caused by these fungi [30]. Studies have shown that melanin of zoopathogenic and phytopathogenic fungi is essential for their parasitizing, due to its antioxidant properties [101].

Melanin pigment extracted from several fungal species has shown the ability to scavenge free radicals (reactive nitrogen and oxygen species), becoming a potential natural antioxidant. Melanins produced by Exophiala pisciphila and Aspergillus bridgeri ICTF-201 exhibited a significant DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity comparable with that of synthetic melanin, indicating its antioxidant potential [102103]. Melanin produced by Schizophyllum commune showed high free radical scavenging activity in a dose-dependent manner, when the melanin concentration was increased from 10 to 50 μg, the scavenging activity was also increased from 87% to 96%, similar to those obtained using ascorbic acid (standard compound used to measure free radical scavenging activity) [34]. Melanin pigment of Fonsecaea pedrosoi has antioxidant potential by reducing Fe(III) to Fe(II), ensuring the balance of its redox chemical microenvironment and minimizing the effect of oxidation of fundamental structures on fungal growth [104]. Similar results were also observed for melanin from Ophiocordyceps sinensis, which proved to be an effective DPPH radical scavenger and a strong ferrous iron chelator [105]. The chelating power of fungal melanin can be explained by various functional groups present in the structure of this pigment, which provide an array of multiple nonequivalent binding sites for metal ions [1422].

It has been reported that substances acting as antioxidants protect cells from ROS-mediated DNA damage, which can result in mutation and subsequent carcinogenesis. The excess free radicals may attack cellular constituents, as the cell membrane, nucleic acid, protein, enzymes, and other biomolecules, by peroxidation, resulting in the severe damage of cell functions and subsequent serious deleterious effects on the organism [106]. It has been reported that melanin protects melanocytes and keratinocytes from the induction of DNA strand broken by hydrogen peroxide, indicating that this pigment also has an important antioxidant role in the skin [107]. Studies in our laboratory showed that melanin extracted from hyperpigment-productive mutant (MEL1) of A. nidulans has the ability to scavenge the biological oxidants, as HOCl, and may be a promising material in cosmetic formulations to protect the skin against possible oxidative damage [31].

There is experimental evidence that fungal melanin may also act as an anti-aging drug, due to its action in reducing the generation of free radicals, clearing away the free radicals produced in excess, and enhancing the activities of antioxidant enzymes. Studies have shown that one of the major causes of aging is the surplus free radicals produced during the oxidative metabolism in the human body [108]. It was demonstrated that the melanin produced by fungus Lachnum singerianum YM296 significantly inhibited the formation of lipid peroxidation products and slowed down the aging process, elevating the levels of superoxide dismutase, glutathione peroxidase, and catalase and decreasing the level of malondialdehyde in mice liver and brain homogenate and serum, suggesting that this pigment could be used as a new anti-aging drug [109].

Researches have also shown that some fungal melanin exhibits immunomodulatory activity through the inhibition of pro-inflammatory cytokine production in T lymphocytes and monocytes, as well as fibroblasts and endothelial cells [12110111]. During an inflammatory response, cells of the innate and acquired immune systems release a variety of mediators, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukins (IL), and the reactive nitrogen and oxygen species, which are implicated in the pathogenesis of a number of inflammatory diseases [112]. [113] reported that treatment of macrophages activated in vitro with melanin from the fungus F. pedrosoi inhibited the production of nitric oxide and Th1 cytokines. The study performed by [114] showed that the expression of inducible nitric oxide synthase gene decreased and lower levels of cytokines, such as IL-12 and TNF-α, were observed when activated macrophages were incubated with melanized cells of the Fonsecaea monophora fungus. Our studies demonstrated that melanin extracted from a highly melanized mutant (MEL1) of Anidulans inhibited NO production in LPS-stimulated macrophages, with a maximum response of 82% inhibition, and also showed a dose-dependent inhibitory effect on TNF-α production, reaching an inhibition of 51.86% at a melanin concentration of 100 μg/mL. These results suggest that melanin from Anidulans has potential as an anti-inflammatory agent and may be used in the future for development of new drugs with therapeutic utility [32].

Some studies have proposed that fungal melanin exhibits anti-radiation activity in vivo and in vivo and then could be explored as a probable radioprotector [16115]. Since melanin has a stable free radical population, it is thought that the radioprotective properties of this pigment result from a combination of physical shielding and quenching of cytotoxic free radicals generated by radiation [18]. [116] showed that Lachnum extracellular melanin (LEM404) had strong anti-ultraviolet radiation activity because the survival rates of Escherichia coliStaphylococcus aureus, and Saccharomyces cerevisiae under UV radiation were significantly increased after in vitro addition of LEM404. Compared with the control groups, the antioxidant defense systems, such as superoxide dismutase and glutathione peroxidase activities, were improved significantly in mice of experiment groups, and the reactive oxygen species detected by malondialdehyde content were decreased significantly. These results confirmed that fungal melanin could be used as component of photoprotective creams mainly for its free radical scavenging rather than its light absorption properties. The probable mechanisms of radioprotection by melanin appear to be modulated in pro-survival pathways, immune system, and prevention of oxidative stress. It was reported that melanin isolated from the fungus G. simplex reduced the radiation-induced overproduction of pro-inflammatory cytokines (IL-6 and TNF-α), which might help in the recovery from radiation injury by preventing the aggravation of inflammation and oxidative stress [33]. This study confirmed the possible use of melanin-coated nanoparticles for protecting against radiotoxicity during radioimmunotherapy [117].

Recent studies have demonstrated that, in addition to the ability of transferring electrons arising under the action of radiation, melanin also possesses ionic conductivity due to its ability to transform any type of radiation energy not only into heat but also use it for the maintenance of redox processes in cells [118]. It was assumed that melanin pigments, participating in redox reactions, are able to perceive the energy of radiation (UV, visible light, and radiation) and convert it into useful reducing power for metabolic processes. This hypothesis is supported by the discovery of melanized fungi in soils contaminated by radioactive nuclides and areas around the damaged Chernobyl nuclear reactors, which not only survive high radiation levels but also have enhanced growth upon exposure [1619119120]. Owing to its semiconductor property, melanin becomes a promising material for organic bioelectronic devices like transistors, sensors, and batteries [121].

Fungal melanins also exhibit growth inhibitory effect against various microorganisms. The extracellular melanin isolated from S. commune showed significant antibacterial activity against E. coliProteus sp., Klebsiella pneumonia, and Pseudomonas fluorescens and antifungal activity against dermatophytic fungi, Trichophyton simii, and Trubrum [34]. The A. auricula melanin displayed inhibitory activity on biofilm formation of the three bacterial strains, E. coli K-12, Pseudomonas aeruginosa PAO1, and P. fluorescens P-3, and there was a proportional reduction in biofilm biomass with the increase in pigment concentration. Confocal laser scanning microscopy (CLSM) analyses showed that the three strains formed thick and compact biofilms when grown in the absence of pigment, but the presence of Aauricula melanin resulted in thinner and looser cell aggregations on surfaces instead of normal biofilm architecture. This study suggested that Aauricular melanin inhibits quorum-sensing (QS)-regulated biofilm formation in all strains tested without interfering with their growth [122]. Silver nanoparticles incorporated Yarrowia lipolytica melanin exhibited antimicrobial activity against the pathogen Salmonella paratyphi, and they were also effective at disrupting biofilms on polystyrene as well as glass surfaces [123]. These nanoparticles displayed excellent antifungal properties toward an Aspergillus sp. isolated from a wall surface, suggesting the application of these nanoparticles as effective paint additives. The melanin-silver nanostructures with broad-spectrum antimicrobial activity against food pathogens also have potential applicability in food processing and food packaging industries [124].

The anti-cell proliferation effect of fungal melanin in tumoral cell lines has already been demonstrated. [34] reported that the extracellular melanin produced by the fungus S. commune was effective against human epidermoid larynx carcinoma cell line (HEP-2) in a concentration-dependent manner, indicating its potential application in cancer chemoprevention and chemotherapy.

The evaluation of the effect of fungal melanin on non-tumor cells is also interesting because it may serve as alternative to acute in vivo toxicity testing, avoiding the indiscriminate use of animals. The melanin produced by A. bridgeri was evaluated in vitro cytotoxicity assay using cell lines TE 355.Sk derived from normal human skin fibroblasts and HEK-293 derived from human embryonic kidney cells, and no cytotoxicity was observed against the two cell lines [103]. In our studies, the toxicity of the melanin from A. nidulans was also evaluated due to its potential practical application as antioxidant and anti-inflammatory agent. The results showed that the viability of mouse macrophages remained greater than 90% when these cells were treated with a high melanin concentration (100 μg/mL), indicating that this pigment has low cytotoxicity [32]. We also showed that the toxicity of Anidulans melanin on mouse fibroblast McCoy cell line, after metabolic activation with hepatic S9 microsomal fraction, was much lesser (CI50 = 413.4 ± 3.1 μg/mL) than known cytotoxic agents such as cyclophosphamide (CI50 = 15 ± 1.2 μg/mL). In this study, we demonstrated that this melanin pigment did not induce gene mutations in different strains of Salmonella typhimurium used in the Ames assay. Based on these results, we suggest that the melanin produced by Anidulans does not cause significant damage to the cellular components and might be used in the future for development of new therapeutic drugs [32].

5. Biotechnological applications of melanin

With the current knowledge about physical and chemical properties and the broad spectrum of biological activities, fungal melanins have attracted growing interest for their potential use in the fields of biomedicine, dermocosmetics, nanotechnology, and materials science.

5.1. Bioelectronic applications

In recent years, the electronics industry has been driven to develop materials and components that are cheaper and more environmentally friendly. As melanin has characteristics of functional materials and bioorganic, a growing number of researchers in the fields of materials science and organic electronics see the melanin with great interest, taking advantage of their properties for applications in organic electronic devices. Melanins present interesting optoelectronic properties, such as high optical absorption in the UV-Vis range, good transmission electronic, and ionic conductivity appreciably, pointing this biomaterial as a promising active component in organic electronic devices with low environmental impact [118121125127].

Among the physical properties of melanin, the electrical conductivity is one of the most interesting to investigate in the perspective of technological application. The electrical conductivity properties of this biopolymer are similar to those of amorphous semiconductor solids, and then it can be considered an organic semiconductor, which is largely available and biocompatible and, consequently, cheaper and easier to process with respect to inorganic semiconductors, as silicon germanium. In particular, it can be considered a promising material for sensors and photovoltaic devices, due to broadband spectral absorbance and charge transport properties [128].

The technical literature describes the integration of organic semiconducting polymers as melanin in silicon electronic devices in view of the possibility of achieving multifunctional systems that combine electrical and optical properties of semiconductors, the structural versatility and mechanical characteristics of materials, and processing polymeric [129]. The production of devices based on thin film melanin exhibited electrical conductivity comparable to that of amorphous silicon [130]. In this study, melanin films showed excellent thermal stability and adhere well to glass substrates and silicon, indicating the possibility of using this technique for the production of films from synthetic melanin. Other groups have published various device architectures with applications such as memory (metal-insulator-semiconductor geometries) [131], batteries [132], and biomimetic interfaces [133].

Deposition of homogeneous melanin layers for optoelectronics application is an issue of considerable technological relevance. Synthetic melanin thin films deposited by spray-coating presented features ascribed to an amorphous semiconducting material [134]. They also showed that further improvement of conductivity together with an increased absorption in the NIR region, by doping the synthetic melanin macromolecule, could make this material a good candidate for optical sensing applications. It has been reported that the iron-melanin coating markedly enhances the catalytic activity of the gold nanoparticles (AuNPs) for both the hydrogen peroxide electroreduction and hydrogen evolution reaction [135]. This strategy may be used to improve nanomaterials with potential applications as efficient catalysts and electrocatalysts. Studies have shown that synthetic melanin-like nanoparticles complexed with paramagnetic Fe3+ ions have potential as a highly efficient and nontoxic contrast agent for magnetic resonance imaging instead of Gd3+-based contrast agents, which can cause nephrotoxicity [136].

The optical and electronic properties of melanin have attracted the attention of researchers for the production of continuous thin films from conventional synthetic melanin, which have been used for a number of different device configurations, including chemi-sensors, next-generation solar cells, and a range of other detectors [126130134]. Potential also exists to use melanin films as an effective radiation sensitizer that could greatly improve the spectral range and efficiency of superconducting transition-edge bolometers [137].

The metal chelation properties of melanin offer interesting possibilities for melanin-based metal ion sensing. A piezoelectric sensor system capable of real-time detection of metal ions was constructed by cross-linking melanin onto the gold electrode of quartz crystal microbalance (QCM) and showed high sensitivity and selectivity to metal ions particularly for Hg(II) [138].

Melanin has many other interesting properties, such as ultraviolet absorption, which has been utilized to prepare optical lenses or filters. Studies have shown that it is possible to use melanin as an ultraviolet, visible and near-infrared absorbing pigment in opthalmic devices, protective eyewear, windows, packaging material, umbrellas, canopies, and other similar media suitable for providing protection from radiation [139140]. The incorporation of the melanin in solid plastic films of polyvinyl alcohol (PVA-melanin film system) to be used in conjunction with other plastics to make laminated sheets or lenses, including sunglasses, ski goggles, ophthalmic prescription lenses, helmets, windows, light filters for artificial lighting, and other light filters that protect people from potentially damaging UV and high-energy visible light has also been reported [141].

5.2. Medical applications

Despite its high biocompatibility, the use of melanin as a novel biomaterial in pharmaceutical and biomedical applications reported in literature is still scarce. A study performed with melanin nanoparticles as biocompatible drug nanocarriers, using metronidazole (antibiotic drug), showed that melanin could be a very interesting nanocarrier drug release device because it strongly responds to pH, being a very interesting feature for the treatment of intestine and colon diseases, which would greatly benefit with pH targeting [142]. Another study showed that systemic melanin-covered nanoparticle (MN) administration reduced hematologic toxicity in mice treated with radiation and that these structures provide efficient protection to bone marrow against radiotoxicity during radioimmunotherapy and in some cases external beam radiation therapy, permitting the administration to tumors of significantly higher doses [117].

Melanin has also been used to treat various types of malignant cancer tumors, disorders of the immune system including AIDS, diseases of blood origin and disorders due to the disturbances in cell homeostasis, and complex and hardly curable mental disorders (schizophrenia, epilepsy) involving nervous and other regulatory systems. A study on the use of melanin for the treatment of Parkinson’s disease, an amelioration in the monkeys’ overall functional ability and secondary motor manifestations by the administration of an effective amount of melanin in monkeys treated with MPTP (1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine), a toxin that causes a neurodegenerative disease, was observed. This study demonstrated that toxin-induced Parkinson’s disease could be prevented in the melanin-treated animals because the administered melanin causes chelation or scavenging of toxins, such as MPTP, thus preventing a neurodegenerative disease, such as Parkinson’s disease. The results of this study also showed that melanin administration to aid the recovery of neurons in a mammal having neuron injury suggests that melanin can be used to treat Alzheimer’s disease [143].

Owing to their ability to increase the permeability of the blood-brain barrier, the melanin is also useful as carriers for other therapeutic agents, which must reach brain tissue to produce their therapeutic responses [144]. Two examples of such therapeutic agents that will cross the blood-brain barrier when linked to melanin are boron and nerve growth factor. According to the same authors, the melanin is also an effective vehicle for the transport of boron to cancerous sites in the body, mainly when the cancerous cells to be treated are located in the brain, because this pigment binds boron very strongly. The melanin can also function as a carrier for nerve growth factor due to the ability to get nerve growth factor across the blood-brain barrier, and this is the major advantage over conventional therapy.

In recent years, efforts have been focused on investigating the potential use of this pigment as active material in tissue repair engineering. Bettinger et al. [145] reported that thin films of melanin were found to enhance Schwann cell growth and neurite extension in rat pheochromocytoma cells (PC12 cells) compared to collagen films in vitro. Melanin films also induced an inflammation response that was comparable to silicone implants in vivo, and the implants were significantly resorbed after 8 weeks. These results showed that melanin thin films have great potential in the reconstruction of tissues, being biodegradable, and possess inflammatory response comparable to silicone. Another study of the biocompatibility of melanin thin films demonstrated that the melanin film effectively supports the growth of undifferentiated stem cells and their differentiation into neuronal precursors and neurons [146]. They related that high-quality melanin thin films display appealing features, such as reversible conductivity by controlled hydration—dehydration steps—excellent biocompatibility with stem cells, and water-resistant adhesion, for bioelectronic applications, e.g., in organic electrochemical transistors (OECTs), which can translate cellular activity into electrical signals [125147]. It has also been reported that melanin thin films possess highly desirable physical and biological properties that make them ideal for organic bioelectronic devices [130].

In cosmetic industry, there are great interests in the melanin, especially to protect against the noxious effects of UV radiation by incorporation in skin photoprotection formulations [35148]. The protective action of melanin is related to its high efficiency to absorb and scatter photons, particularly the higher-energy photons from the UV and blue part of the solar spectrum. Very likely, melanin photoprotection is also due to its ability to quench excited states of certain molecules and scavenge ROS that may be generated in pigmented cells [126]. Development of methods for producing melanin soluble in aqueous cosmetic buffers at physiological pH and temperature may make possible the use of this pigment as ingredients of face and hand creams, lotions, antiaging ointments, or foundation makeups, acting as a screen and antioxidant for the protection against photoinduced skin damages [149]. Other dermocosmetic applications of melanins include the use of the pigment for hair dyeing and the development of novel strategies for hair recoloration [150].

Since melanin has a stable free radical population, it is thought that the radioprotective properties of this pigment result from a combination of physical shielding and quenching of cytotoxic free radicals generated by radiation [18]. Some studies suggest the possible use of melanin-coated nanoparticles in medicine, mainly for protecting patients against the harmful effects of gamma rays during radioimmunotherapy [34151]. Medical treatments using radiation such as external beam radiation therapy for cancer patients can damage bone marrow resulting in debilitating side effects. In experimental models, melanin can successfully shield bone marrow from such side effects. Mice treated with melanin-coated nanoparticles have higher white blood cell and platelet counts than control mice after radiation treatment [117]. It has been reported the use of melanin, a biopolymer with good biocompatibility and biodegradability, intrinsic photo-acoustic properties, binding ability to drugs, and chelating property to radioactive metal ions, as an efficient endogenous nanosystem for imaging-guided chemotherapy [152]. According to the authors, melanin nanoparticles could successfully enter into the tumor and act as an efficient drug-delivery system, thereby greatly increasing the safe utility of the drugs for tumor treatment and significantly lowering the dosage used and its side effects.

A valuable biotechnological approach to the melanin-mediated synthesis of silver nanostructures with broad-spectrum antimicrobial activity has been developed. Silver nanostructures synthesized with melanin derived from Y. lipolytica displayed excellent antifungal activity against an Aspergillus sp. isolated from a wall surface, indicating its potential application as effective paint additives [123]. The melanin-mediated nanostructures with broad-spectrum antimicrobial activity against food pathogens may be considered suitable for many practical food packaging applications because they can effectively inhibit the growth of pathogens and increase the shelf life of packed food products [124].

5.3. Environmental applications

The chemical structure of melanin presents many oxygen-containing groups, including carboxyl, phenolic and alcoholic hydroxyl, carbonyl, and methoxy groups, which have the ability to bind to a broad spectrum of substances [153]. In literature, studies have confirmed that fungal melanin acts as metal chelators, enhancing the biomass-metal interaction and consequently its biosorption capacity [14]. Study conducted by [154] showed that a melanin-rich strain of the fungus Cladosporium cladosporioides biosorbed 2.5- to 4-fold more Ni, Cu, Zn, Cd, and Pb ion than non-melanic Penicillium digitatum. These authors also studied the culture of Ccladosporioides in different growth times and found that a culture grown for two days is not pigmented and has only 34% of Cd adsorption rate that obtained for pigmented biomass after 4 days of growth [155]. Another study reported that melanized fungus Armillaria adsorb high concentrations of cations from the surrounding environment; some ions (Al, Zn, Fe, Cu, and Pb) were 50–100 times more concentrated on rhizomorphs than in soil [156]. The results obtained in our laboratory using a melanin-overproducing mutant (MEL1) from A. nidulans fungus [3193] showed that biosorption capacity for neodymium and lanthanum varied with stage of growth of this mutant; the biomass obtained after 72 hours of growth exhibited a 75% increase compared to the biomass of 48 hours. This result is related to melanin production during growth of the MEL1 mutant, since the biomass 48 hours is slightly pigmented, while the 72 hours biomass is dark due to the increased production of pigment [157]. Therefore, the pigmented biomass of the MEL1 mutant may be considered as a promising biosorbents for removal/recovery of the rare earth elements from wastewater due to the presence of the melanin increase significantly metal complexing capacity, improving the efficiency of biosorption process [157].

Some melanized fungi have shown to be good candidates for bioremediation of contaminated sites, due to the ability of fungal melanin to bind to heavy metals and radionuclides in contaminated sites. Experimental evidence shows that the accumulation of 90Sr by conidia or mycelium by a range of microfungal species is greater in pigmented than in unpigmented species [158]. [159] In a study on the uptake efficiency of the radiocesium (137Cs) and radiocobalt (60Co) in melanized and nonmelanized fungi, it was observed that 60% of both radionuclides were uptaken by melanin of A. alternata and Aspergillus pulverulents. These results can be explained by melanin or other natural pigments present in the cell wall of these fungi that can act as the radiation receptor and/or as an energy transporter for metabolism. Other studies have demonstrated the potential application of the melanized fungi for the removal of radionuclides and heavy metals from aqueous solutions, providing an alternative means to affect cleanup of industrial effluent [16120160164]. It has been reported that fungal melanin arranged in nanoparticles protects against extremely high levels of ionizing radiation and suggests that the protective efficacy of this pigment is a function of its chemical structure, the presence of stable free radical, and spatial arrangement [18]. According to the authors, these nanoshells have the potential use for environmental bioremediation, for example, to prevent the spread of radioactive contamination to ground water because the melanin is expected to encapsulate the radioactive particles and thereby reduce their spread. In this way, melanin nanoshells may be used to contain radiation from radioactive waste and biomedical radioactive materials.

6. Conclusion

Melanin possesses physicochemical properties and biological activities that make it a suitable biomaterial for a wide range of applications in cosmetic, pharmaceutical, electronic, and food processing industries. In addition, this pigment has a considerable interest biotechnological because it can be produced on a large scale with low cost, making its use for future practical applications economically advantageous. However, it is necessary to expand the knowledge about the structure-property-function relationships for the development of melanin-based technology. In the context, we hope that the information in this book will be useful and will encourage a greater number of researches on fungal melanin, which might be useful to deploy innovative and sustainable solutions for human health and the environment.

Continue reading Production of Melanin Pigment by Fungi and Its Biotechnological Applications

Human Helminths. Friend or Foe?

Chapter 87 Helminths: Pathogenesis and Defenses

Helminth Genomics: The Implications for Human Health

Circa 2009:  More than two billion people (one-third of humanity) are infected with parasitic roundworms or flatworms, collectively known as helminth parasites.

LET THAT SINK IN…Or maybe not…8)

These infections cause diseases that are responsible for enormous levels of morbidity and mortality, delays in the physical development of children, loss of productivity among the workforce, and maintenance of poverty. Genomes of the major helminth species that affect humans, and many others of agricultural and veterinary significance, are now the subject of intensive genome sequencing and annotation. Draft genome sequences of the filarial worm Brugia malayi and two of the human schistosomes, Schistosoma japonicum and S. mansoni, are now available, among others. These genome data will provide the basis for a comprehensive understanding of the molecular mechanisms involved in helminth nutrition and metabolism, host-dependent development and maturation, immune evasion, and evolution. They are likely also to predict new potential vaccine candidates and drug targets. In this review, we present an overview of these efforts and emphasize the potential impact and importance of these new findings.

Conversely – Further Reading

Parasites – Centers for Disease Control

DATA- Parasites – Soil-transmitted helminths

The Global State of Helminth Control and Elimination in Children

Soil-Transmitted Helminthiasis in the United States: A Systematic Review—1940–2010

Public health deworming programmes for soil-transmitted helminths in children living in endemic areas.

Modern Pellagra

Recognising the return of nutritional deficiencies: a modern pellagra puzzle

A 34-year-old previously well woman presented with a 4-week history of diffuse erythema and crusting of skin affecting all four limbs. Examination revealed erythematous skin plaques associated with ulceration and fissuring affecting sun-exposed areas of all four limbs primarily on the dorsal surfaces, and a body mass index of 17 kg/m2. She was admitted under the infectious diseases unit, and an autoimmune and infective screen was performed which returned unremarkable. Dietetic consultation led to the diagnosis of severe protein-energy malnutrition, consequent to a severely restricted, primarily vegan, diet. Analysis of the patient’s reported diet with nutritional software revealed grossly suboptimal caloric intake with risk of inadequacy for most micronutrients, vitamins and minerals, including niacin[B-3]. Oral thiamine[B-1], multivitamin, iron supplementation and vitamin B complex were started, and a single intramuscular vitamin B12 dose was administered. Marked improvement was seen after 6 weeks, with near-complete resolution of skin changes. These findings supported a diagnosis of pellagra.

“Our daily bread” – the scourge of pellagra

Meera Ladwa
London, England, United Kingdom

In the northern Italian town of Ferrara hangs a little-known painting by Giuseppe Mentessi (1857-1931). Surrounded by a field of maize, a woman carries her exhausted child in her arms, her eyes downcast with suffering. Behind this painting lies a story of medicine, food, economics, and culture – the story of pellagra, perhaps one of the greatest tragedies of malnutrition known to the Western world.

Exhibited at the Venice Biennale in 1895, Mentessi’s image of a cornfield depicts what once was a common sight throughout the region. Maize or corn was first domesticated as a cereal crop in the Americas, making its way to Europe via traders in the sixteenth century.1 This new foodstuff proved to be a lifeline for Italian agricultural workers who labored under the pressures of wheat shortages and insecure employment.2 With its low cost and high yield, cornmeal or polenta was quickly established as the staple food of the poor. Landowners were eager to profit from maize production, and eventually forests, vineyards and pastures across the Veneto and Lombardy were replaced by fields of this single crop. It was fitting that Mentessi named his picture “Our Daily Bread” – for by the 1800s, northern Italian peasants were living on a diet made up almost exclusively of corn.2

At the same time, increasing numbers of them were falling victims to a new disease. Characterised by a flaking rash on the sun-exposed skin of the arms and neck, they named it pellagra (from pelle agra, loosely translated as “rough skin”).3  Symptoms included confusion, mania, lethargy, and eventual death. While the link between pellagra and maize dependence was quickly recognized by scientists at the time, the mechanism remained a mystery. Today, we know that pellagra is a disease of severe niacin (vitamin B3 or nicotinic acid) deficiency. Niacin is the precursor of two essential coenzymes of cellular activity – nicotinamide adenine dinucleotide (NAD) and NAD-phosphate (NADP) – both involved in DNA repair, cell signalling, and metabolism.4 Tissues with high energy requirements and high cell turnover are particularly vulnerable to their deficiency. Without sufficient dietary niacin – or tryptophan, the amino acid used in its biosynthesis – a systemic disease occurs affecting the skin, gastrointestinal tract, and nervous system. The resulting clinical features have been famously described as the 4 D’s: dermatitis, diarrhea, dementia, and death.4

The unvarying cornmeal diet that poor Italians relied on in the lean winter months, without the addition of vegetables, dairy products, or meat, was severely deficient in both niacin and tryptophan.

Individual cases of the deficiency disease are occasionally found in chronic alcoholics, elderly widowers who have an improper food intake, and inpatients suffering from malabsorption. ~[WHO]Pellagra, its Prevention and Control in Major Emergencies.

An epidemic of pellagra resulted, with thousands of deaths attributed to the illness – particularly in women, who had increased nutritional needs because of pregnancy and breastfeeding, and whose inferior social position meant that they tended to have less food then the men in their family.2 So common did the neuropsychiatric effects of the illness become, that mental asylums such as Venice’s San Servolo and San Clemente were full of victims of what was then termed “pellagrous insanity.”3 For almost two centuries, pellagra was endemic in the agricultural lands of the Po valley across northern Italy.1

It was this widespread suffering which Giuseppe Mentessi sought to depict in his painting. Born into poverty in Ferrara, Mentessi went on to have a successful career as an art teacher and professor at the Accademia di Brera in Milan.5 Nevertheless, his work maintained a deep affinity with his humble origins and he often used his art to highlight the social issues of the day. In “Our Daily Bread,” he shows the misery of the Italian rural poor, with the woman’s sickly countenance a mark of the effects of pellagra. What he depicted in paint, the writer Goethe described in words in his ‘Italian Journey:’ “Of the (Italian) inhabitants, I have little to say and that unfavourable … (the) sallow complexion of the women spoke of misery and their children looked just as pitiful …  I believe that their unhealthy condition is due to their constant diet of yellow polenta …”6

It was not the diet of cornmeal alone which gave rise to pellagra in southern Europe, but also the method of preparation. While maize had been a staple food of central America for thousands of years, the indigenous peoples were accustomed to soaking the dried corn kernels in alkaline lye or quicklime before cooking. This process, known as “nixtamalization,” increased the bioavailability of bound niacin in the corn by converting it into the water-soluble free compound, allowing it to be absorbed by the gut.7 As a result of their traditional cultural methods of preparation, the native people of the Americas did not suffer from pellagra.

When corn was brought across the Atlantic to Europe, the tradition was lost. Furthermore, pellagra was then introduced to the Americas, where European colonizers grew and ate corn without realizing the benefit of nixtamalization or the importance of a varied diet. Particularly in the Southern states, in the economic downturn following the American Civil War, the daily fare for poor people consisted almost entirely of corn-based products such as cornbread and grits. Rural sharecroppers and populations lacking access to fresh produce – in prisons, coal-mining camps, and cotton-mill towns – were particularly vulnerable to niacin deficiency. The devastation occurred on a grand scale; across the United States from 1906 to 1940 approximately 3 million cases and 100,000 deaths were attributed to pellagra.8

It was a US public health physician, Joseph Goldberger, who determined that pellagra was a nutritional deficiency and not (as was commonly supposed) an infectious epidemic. In 1915, he carried out a series of experiments in Mississippi prisoners which demonstrated that symptoms of pellagra appeared after six months of eating only corn-based foods; when fresh, varied produce was introduced, the illness resolved. He concluded that “no pellagra develops in those who consume a mixed, well-balanced diet,” yet struggled to convince the political establishment that poor social conditions might be responsible for the disease.9 It took several more decades of research – including the isolation of niacin itself from liver tissue by Conrad Elvehjem in 1937 – before federal recommendations to fortify flour supplies with vitamins led to the eradication of the condition in the United States by 1945.7

In Italy, economic growth helped end the pellagra epidemic during the 1950s, but not before the illness had provoked widespread debate about the impact of social injustice and deprivation on human health, with commentators such as Flarer (1849) referring to pellagra as “malattia del padrone” – “illness due to the landlord.”1 Today, we understand more than ever the impact of socio-economic factors on our health, with recognition that the West’s adoption of a highly-processed, energy-dense diet has contributed to our modern epidemics of obesity and diabetes. Such “malnutrition in the midst of plenty” has echoes of Mentessi’s work. It is said that he had the idea for his painting when he was taking an afternoon walk through a field of corn and was struck by the contrast between the bountiful crop and the sickly peasant woman with her child: “misery, perhaps hunger, in the middle of that insolent and healthy wealth!”10 In both Europe and the United States, it is still the poorest sections of society who suffer the most from their dependence on an abundance of cheap calories. While it may have been largely forgotten, the story of pellagra ought to be a lasting lesson in how social structures, globalization, and economic change once conspired to cause thousands of deaths from an entirely preventable disease.


Paragonimiasis is a zoonotic infection caused by a highly evolved parasite with a complex life cycle that includes at least three hosts. Two intermediate hosts, a snail and a crustacean, and a mammalian definitive host are necessary to complete the life cycle of this parasite. Human and animal infections result from the consumption of raw or undercooked crustaceans (i.e., the second intermediate host) or through consumption of raw or undercooked meat from a paratenic host. Paragonimus is the only human parasite where the adults reside in the human lung. The genus Paragonimus is highly successful and is endemic in Asia, Africa, and the Americas. Paragonimus kellicotti is the species endemic to North America. Physicians in countries without endemic disease may encounter patients with paragonimiasis who have immigrated with the disease or who contracted the disease while traveling or through ingestion of imported contaminated food. Many infections are subclinical and cause only mild disease. Sometimes, however, severe infections occur and may be fatal. Two of the five patients with P. kellicotti infections that have been reported over the past three decades were associated with relatively severe disease. These patients required surgery, with one requiring a second course of praziquantel. The most recently reported patient had a remote P. kellicotti infection, but the residual pleural effusion caused by the Paragonimus infection became infected by bacteria and directly contributed to his demise.

The diagnosis of paragonimiasis should be considered when the appropriate clinical signs and symptoms, radiologic findings, and food history are supportive. Cough and hemoptysis are the most common presenting symptoms in patients with paragonimiasis. Diagnosis may be achieved through the demonstration of the characteristic operculate eggs in respiratory secretions, stool, or tissue biopsy specimens. Immunodiagnostics are excellent tools for assisting with the diagnosis of imported paragonimiasis, particularly for patients who do not have demonstrable eggs in clinical specimens. The utility of immunodiagnostics for detecting infections caused by P. kellicotti remains to be determined. Praziquantel is an effective treatment, but surgery may be necessary for patients with complicated pleural disease or cerebral paragonimiasis. Control of paragonimiasis in animals is impractical because of wild animal reservoirs, but in humans infections may be averted through the avoidance of certain foods and by thorough cooking. Crayfish in North America may harbor P. kellicotti and therefore should be thoroughly cooked prior to consumption.

Sleep deprivation, dopamine and addictive behaviors

For a number of years now science has born out linkages between sleep deprivation, compulsive actions and addictive behaviors.

I googled, “sleep deprived hedonic desires” and was met with many first quality links. The following are a handful from the first page of search results.

Be Easy! – mJL

why do statins work and what on?

Statins work because they are antimicrobial. Stopping the microbial process results in there not being what they call cholesterol from being used to repair the microbial damage. We also call it scar tissue.

1. Bacteria
2. Archaea
3. Fungi
4. Protazoa
5. Algae
6. Viruses

If you have use for statins in your body it is because you have one, some or all of these overgrowing in or on your person. So not really a waste of time if you have one of these living in or on you.

Just sayin…Statins might be the lesser of the evils.

Unexpected antimicrobial effect of statins

Is There Potential for Repurposing Statins as Novel Antimicrobials?

How do statin drugs affect aging?