Palmitoylethanolamide: A Natural Body-Own Anti-Inflammatory Agent, Effective and Safe against Influenza and Common Cold

Palmitoylethanolamide (PEA) is a food component known since 1957. PEA is synthesized and metabolized in animal cells via a number of enzymes and exerts a multitude of physiological functions related to metabolic homeostasis. Research on PEA has been conducted for more than 50 years, and over 350 papers are referenced in PubMed describing the physiological properties of this endogenous modulator and its pharmacological and therapeutical profile. The major focus of PEA research, since the work of the Nobel laureate Levi-Montalcini in 1993, has been neuropathic pain states and mast cell related disorders. However, it is less known that 6 clinical trials in a total of nearly 4000 people were performed and published last century, specifically studying PEA as a therapy for influenza and the common cold. This was done before Levi-Montalcini's clarification of PEA's mechanism of action, analyzing the role of PEA as an anti-inflammatory agent. We will review in depth these studies, as the results support the effectiveness and safety of PEA in flu and respiratory infections.

Introduction
Palmitoylethanolamide (PEA) is a food component known for more than 50 years. PEA is synthesized and metabolized by different animal cell types and also present in plants. It exerts a multitude of physiological functions related to metabolic and cellular homeostasis. PEA was already identified in the 50s of the last century as a therapeutic substance with potent anti-inflammatory properties. Since 1970, the anti-inflammatory and other immune-modulating properties of PEA have been shown in a number of placebo-controlled double-blind clinical trials on influenza and common cold. Positive results coincided with the clinical use of PEA in former Czechoslovakia under the brand name Impulsin.
Since 2008, PEA has been marketed as a food for special medical purposes in Italy and Spain, under the brand name Normast (Epitech Srl). Recently, a food-supplement named PeaPure was introduced (JP Russell Science Ltd.). In the USA, PEA is under evaluation as a nutraceutical for inflammatory bowel syndrome (proposed brand name Recoclix, CM&D Pharma Ltd.; Nestlé).
Research on PEA has been conducted since its discovery and over 350 papers are referenced in PubMed describing its physiological properties and role as endogenous modulator as well as its pharmacological and therapeutic effects. PEA is an interesting anti-inflammatory therapeutic substance and might also hold great promise for the treatment of a number of (auto)immune disorders, including inflammatory bowel disease and inflammatory diseases of the CNS. In this paper, we will review the role of PEA as an anti-inflammatory agent and as potential treatment for influenza and the common cold. The main purpose is to highlight and discuss these earliest findings, including the 6 double blind studies in these indications published in the last century using Impulsin. Although nearly forgotten, these findings could offer new insights or perhaps even alternative options in the light of 2 International Journal of Inflammation the intense debate around the efficacy and safety of the oseltamivir and zanamivir In the present paper, we will discuss the evolution of knowledge on the anti-inflammatory activity of PEA and its effects in the treatment of respiratory infections.

The Early Years: Anti-Inflammatory Activity of Egg-Yolk Based on a Lipid-Fraction
The protective and anti-inflammatory effects of PEA can be traced back in the literature to 1939 [1]. The American bacteriologists Coburn and Moore demonstrated in that year that feeding dried egg yolk to underprivileged children living in poor parts of New York City prevented the recurrence of rheumatic fever, in spite of repeated attacks of hemolytic streptococcal infection. After 1939, Coburn et al. studied 30 children at a convalescent rheumatic home and prescribed four egg yolks daily. No other change in diet was made and no antibacterial drugs were given. Twenty-two of these children contracted 24 serologically positive group-A streptococcal infections, but none showed clinical evidence of rheumatic recurrences. This was in sharp contrast to previous experience in the convalescent home where rheumatic recurrences had been frequently seen each year [2].
Subsequently in 1954, Coburn and colleagues were also the first to report a phospholipid fraction prepared from egg yolk that showed antiallergic activity in an assay in the guinea pig [3].
The antiallergic factor of egg yolk was then purified by Long and Martin in 1956 in such a way that it was clear that this factor showed a biological and chemical similarity to a preparation obtained earlier in 1950 from peanut and what appeared to be a closely related substance described as "vegetable lecithin" [4,5].
The birth year of PEA was 1957. Kuehl Jr. and coworkers reported to have succeeded in isolating a crystalline anti-inflammatory factor from soybean lecithin and they identified it as N-(2-hydroxyethyl)-palmitamide [6]. They isolated the compound also from a phospholipid fraction of egg yolk and from hexane-extracted peanut meal. The product obtained was tested positively in a local passive joint anaphylaxis assay in the guinea pig. When applying their isolation procedure to soybean lecithin, they obtained a partially purified fraction from which the homogeneous factor was obtained by crystallization from cyclohexane. The crystalline material had a melting point of 98-99 ∘ C and was described as neutral, optically inactive, and possessing the chemical formula C18H37O2N.
Hydrolysis of the factor resulted in palmitic acid and ethanolamine and thus the compound was identified as N-(2-hydroxyethyl)-palmitamide. In order to close the circle of isolation and identification, Kuehl et al. were able to synthesize the compound by reflux in ethanolamine with palmitic acid according to a well-known procedure described in the chemical literature of that time. Kuehl et al. further analyzed the anti-inflammatory activity of a series of derivatives of PEA and could prove that the basic moiety of the molecule was responsible for its anti-inflammatory activity. The nature of the acid group appeared to them to be of no importance because in addition to ethanolamine itself, N-(2hydroxyethyl)-lauramide, S-(2-hydroxyethyl)-salicylamide, and N-(2-hydroxyethyl)-acetamide all had potent antiinflammatory properties. These pharmacological properties of the ethanolamine-derivatives appeared to be quite specific since other homologs did not show a biological response in the assay.

The Protective Effects of ''Proto-PEA'' in Streptococcal Infections
Coburn was dedicated to find the cause and prevention of rheumatic fever [7]. He presented his hypothesis that eggs contained an important protective factor against infection, especially in rheumatic fever, in 1960 in the Lancet [2]. He argued that (a) inadequate nutrition is part of a poor environment; (b) rheumatic-fever children usually lack sufficient eggs in their diets; (c) the escape from poverty is followed by an increase in the consumption of eggs and a decrease in the incidence of rheumatic fever; (d) supplementation of children's diets with egg yolk or certain fractions thereof is followed by decreased rheumatic susceptibility; and (e) there is a fraction of egg yolk, which in extremely small amounts has been found to have high antiallergic activity in laboratory animals.
Coburn described his field studies in great detail [2]. Some of these findings are summarized below.
In Field study number 1, = 89, rheumatic boys and girls living at home in New York City all received egg-enriched food; no prophylactic drugs were given. Sixty children had extra eggs during winter and spring months, and 29 served as "controls. " Results were as follows: of the 29 children on their normal diet (with many nutritional deficiencies) 11 had a recurrence. Of the 35 children whose normal diet was enriched with two eggs daily, a quart of milk, meat, butter, and halibut-liver oil, 3 had a recurrence. Of the 25 children whose normal diet was reinforced only with powdered egg yolk (equivalent to six eggs daily), only 1 had a recurrence.
Field study number 2, = 56, was a two-year study into the effect of giving egg-yolk powder (equivalent of four egg yolks daily) to rheumatic children for three to four weeks after they developed hemolytic (group A) streptococcal pharyngitis. No other treatment was given during this period. Results were as follows: of 28 receiving the supplement, only 1 showed fresh rheumatic activity, whereas among 28 "controls, " receiving no supplement, 10 children had fresh rheumatic activity.
Field study number 3, = 40, was a one-year study in which about 40 rheumatic children (with many dietary deficiencies) received a daily supplement of only the protein fraction from four egg yolks. Results were as follows: study was discontinued because of too many rheumatic recurrences.
Field study number 4, = 45, was a four-year study (Chicago, period 1952(Chicago, period -1956 in which a normal (nutritionally deficient) diet of rheumatic children was reinforced with eggyolk alcohol-soluble material (A.S.M. from Wilson Laboratories). No other changes in their inadequate diets were made; no sulphonamides, antibiotics, or other significant drugs were administered. Forty-five highly susceptible rheumatic children received this supplement throughout the school year from September to July. The equivalent of 3 egg yolks was consumed, in the form of an elixir taken twice daily. All but one of these rheumatic children was under fifteen years of age. Results were as follows: a minimum of 17 attacks was expected among them after streptococcal infections, but only 5 occurred.
Coburn concluded that "The data obtained under these various conditions, both in New York and a decade later in Chicago, were found to be statistically significant. " However, he himself acknowledged that all studies had methodological weaknesses [2].
Coburn discussed various experimental findings around that time supporting the idea that there is at least one antiinflammatory substance present in egg-yolk alcohol-soluble material, which was not present in the protein or acetonesoluble material [5,8]. The anti-inflammatory activity was confirmed by different groups, for instance, by measuring joint and skin lesions in either the Arthus or tuberculin reaction. Various models were used and all results were supportive of Coburn's observations. The anti-inflammatory compound clearly was part of the lipid fraction of the egg and not the protein-water fraction.

Acceptance of PEA's Anti-Inflammatory Effects
Already in 1965 the anti-inflammatory activity of PEA seemed to be quite well known in the scientific community. Amongst others, Bachur, from the Laboratory of Clinical Biochemistry and Experimental Therapeutics Branch, National Heart Institute, National Institutes of Health, Bethesda, MD, USA, and colleagues referred extensively to the findings of Kuehl et al. (1957): "Kuehl et al. have previously reported the isolation of PEA, as a naturally occurring anti-inflammatory agent, from egg yolks. PEA was known to occur in nature and to have pharmacological activity" [9]. The group of Bachur analyzed the content of PEA and found it to be present in several tissues of the rat and guinea pig. The amounts found in liver were quite variable, but PEA was consistently found in brain, liver, and muscle tissue and was not detected in other tissues examined. Around that time, the anti-inflammatory action of PEA could also be demonstrated in a classical anti-inflammatory model, the carrageen-induced edema model [10].
In the beginning of the 70s, the modifying effects of PEA on immunological reactions were well established [11]. Perlik et al. summarized [12] that "It has been shown that N-(2hydroxyethyl)-palmitamide (PEA) can decrease the intensity of several inflammatory and immunological processes. " However, between 1958 and 1969 the interest in this compound had apparently decreased, as the same authors stated that "Recently the interest on biological properties of PEA has been revived because of its capacity to increase nonspecific tolerance to several bacterial toxins. "

PEA: Anti-Influenza and Anticommon Cold
New interest arose by the end of the 60s, due to the fact that SPOFA United Pharmaceutical Works brought PEA on the market in 300 mg tablets under the brand name Impulsin to treat influenza and common cold. Different clinical trials supported the effectiveness and safety of PEA for this indication. Most probably the PEA in Impulsin was not specifically formulated but details are not available.
In the period of 1969-1979, the results of a total of 5 trials in adults and one trial in children were published. All of these were double-blinded and placebo-controlled.
In the 1974 paper by Masek et al., the first two doubleblind controlled trials were described with a total of 1344 healthy subjects randomized (see Table 1: Masek 1972a and Masek 1972b). There were in total 40 dropouts during the studies, meaning that 1304 subjects completed the trials. The goal of these two trials was to evaluate the prophylactic and treatment efficacy of Impulsin in upper respiratory tract infections. Both trials ended in February 1973 [13].
The first trial (Masek 1972a) was a treatment trial; 468 employees of the Skoda car factory were randomized in this trial; of these 444 were completers, available for analysis. The employees had to register the following symptoms: temperature of 37.5 ∘ C or higher, headache, sore throat, myalgia, nasal stuffiness or discharge, productive or dry cough, malaise, and fatigue and had to make a clear impression of being sick. Dosing was 600 mg Impulsin three times daily during 12 days (total daily dose 1800 mg PEA).
The second trial was a prophylactic trial; 918 volunteers between 18 and 20 years of age from an army unit were included, and 899 completed the trial period. In this trial, medical personnel registered the complaints during a period of 8 weeks. The treatment schedule was 600 mg PEA three times daily for the first 3 weeks, after which a continuation phase started based on a single dose of 600 mg once daily for 6 weeks.
The results from the first trial showed that patients receiving PEA had a lower number of episodes of fever, headache, and sore throat, compared with placebo patients (18 versus 33). PEA had less effect on symptoms such as nasal stuffiness, discharge, and cough. The episodes of fever and pain were significantly reduced by 45.5% in the PEA group compared with the placebo group ( < 0.05). The beneficial effect of PEA was apparent from the second week of the trial. The total number of sickness days was also significantly reduced in the PEA group. In the prophylactic trial, Masek 1972b, the incidence of disease in the PEA group was 40% lower at week 6, and 32% lower at week 8 compared to placebo ( < 0.0005).
In order to verify the conclusions, 3 more trials in soldiers were conducted. Soldiers were selected as they were housed close together. In the period of 1973-1975, these new trials were initiated (Kahlich 1973(Kahlich , 1974(Kahlich , and 1975 in Table 1) and the results were reported in 1979 by Kahlich et al. [14]. Due to the positive effects, it was felt to be unethical to randomize 1 : 1 and in the last two trials a different randomization schedule was selected, in order to dose the majority of volunteers with Impulsin (2 : 1). The authors compared the incidence of clinical endpoints and the titers of influenza viruses between both the PEA and placebo groups. In all three trials, the soldiers in the PEA group had significantly less symptoms and were less often diagnosed as flu patients (see Table 1). The evaluation of results according to morbidity, regardless of etiology, showed a significant reduction in acute respiratory diseases (ARD) after administration of PEA. In the 1973 trial (901 volunteers), 22.7% of ARD cases were found in the PEA group compared to 34.4% in the placebo group ( < 0.0002). The relevant values in the 1974 trial (610 volunteers) were 19.7% and 40.7% ( < 0.002) and in the 1975 trial (353 volunteers) 10.6% and 28.8% ( < 0.004) [14].
In all studies, Kahlich et al. studied serology in order to document the influenza strains. The codes of these strains are described below; however, the nomenclature is outdated. A fourfold increase in the antibody titer was taken as evidence for infection.
In the 1973 study, serum was obtained from 358 persons. 6.9% of the subjects experienced influenza A 2 E in the PEA group and 18.7% of the subjects in the placebo group ( < 0.005). The manifestation rate (MR), expressing the proportion of sick persons out of all sensitive subjects with serologically proved infection, was 15.4% in the PEA group and 44.9% in the placebo group ( < 0.0002).
In the study of 1974, sera of 108 subjects were analyzed. In the PEA group 3.8% of the subjects suffered from the influenza B Hong-Kong and 21.4% of the subjects in the placebo group ( < 0.01). The MR was 14.3% in the PEA group and 57.1% in the placebo group ( < 0.001).
In the study of 1975, with serum gathered from 212 subjects, only 4.3% of the subjects in the PEA group had influenza A Port Chalmers and 7% of the subjects in the placebo group (nonsignificant difference). The incidence rate of influenza A 2 England was 15.4% in the PEA group and 44.9% in the placebo group ( < 0.0002).
All these clinical trials pointed in the same direction that PEA has clear treatment effects in respiratory infections, can be used as influenza-prophylaxis, and is safe in its use. Side effects were not reported, and Kahlich et al. explicitly stated that "No side effects were registered after several years of clinical testing of Impulsin in military and civilian communities. " Kahlich et al. also pointed out that the effects of PEA had a clear advantage over vaccines and antiviral compounds such as amantadine, due to the optimal balance of efficacy and side effects of PEA. They also stated that the ease of application of PEA offers the possibility to have a quick therapeutic answer ready in case of a flu epidemic, especially in cases of a mismatch between circulating strains and the recommendations from WHO.
A last placebo-controlled study with PEA in children aged 11 to 15, examining the incidence of acute respiratory tract infections, was performed in January 1976 [15]. 457 children were included and divided into 2 groups; 64 children dropped out. In the PEA group, 169 children completed the study who received 300 mg PEA 2 times daily with an interval of 6 hours. The placebo group included 224 children receiving 2 placebo tablets following the same regime as the PEA group.
Blood samples were taken before the study and 8 weeks later in 65% of all children. After 8 weeks, children treated with PEA had 15.7% fewer acute respiratory tract infections than the control group. In children from 11 to 13 years of age, the difference was even more pronounced: 25.5%. Due to the short duration of the intake of PEA and the absence of epidemic influenza during the trial period, the differences between both groups were not very large, and thus no significance was reached.
Taken together, in the period between 1972 and 1977 in total 3627 patients and volunteers completed 6 different placebo-controlled double-blind trials of which 1937 received PEA up to 1800 mg/day. Relevant side effects were not reported and especially the trials conducted during the flu season demonstrated a treatment, as well as a prophylactic effect. The last study in children was not significant due to the fact that during the study period no influenza epidemic occurred.

PEA: Anti-Inflammatory Actions and Its Mechanism of Action via PPAR-Alpha Agonism and Other Targets
Since a decade, NAEs, both as saturated fatty amides (such as PEA) and as poly-unsaturated forms, are found to play an important physiological role in the modulation of immune reactions in a number of autoimmune disorders via a number of different receptors. For instance, Celiac disease is an autoimmune disorder of the small intestine caused by a reaction to gliadin, a gluten protein found in wheat. Most International Journal of Inflammation 5 likely endocannabinoids play here an important modulating role [16]. Anandamide and PEA concentrations in Celiac disease were significantly elevated (100% and 90%, resp.) during the active phase, as was the number of CB1 receptors. The levels returned to normal after remission with a glutenfree diet [17]. This clearly can be interpreted as the activation of a self-repairing mechanism.
In an elegant study on the anti-inflammatory and proapoptotic activities of anandamide, it was shown that it can inhibit tumor necrosis factor--induced NF-B activation [18]. The NF-B inhibitory activity of anandamide was independent of CB1 and CB2. Structure-activity relationships demonstrated that analogs with saturated fatty acyl groups were more active than unsaturated analogs. Saturated acylethanolamides such as PEA therefore offer a new opportunity to modify chronic inflammation in autoimmune disorders.
For a long period of time after the first description of PEA, its mechanism of action remained unsolved, and this led to a weaning interest in the compound after the set of publications on the efficacy and safety of PEA in respiratory infections and flu (in the period of 1970-1980). New interest in the mechanism of action of PEA emerged only after the work of the Nobel laureate Professor Rita Levi-Montalcini, who published a seminal paper in 1993, opening the door to a new understanding of PEA's anti-inflammatory and analgesic actions [19,20]. Starting with her work, it became clear that PEA regulates many pathophysiological processes, and PEA has since been found to be effective in a number of animal models for inflammation, neuroinflammation, neurotoxicity, and chronic pain. Levi-Montalcini highlighted the importance of activation of inflammatory cascades via the activation of nonneuronal cells, such as the mast cells [21]. PEA reduces mast cell migration and degranulation and reduces the pathological overactivation of these cells [21]. Mast cells shift from activated immune-to resting phenotypes under influence of PEA. PEA further reduces the activity of the proinflammatory enzymes, cyclooxygenase, and endothelial, and inducible nitric oxide synthases. PEA has an additional number of other pharmacological and physiological properties, such as its affinity for the novel orphan cannabinoid receptors GPR55 and GPR119 and for the vanilloid receptor TRPV1, as well as for the nuclear peroxisome proliferator-activated receptor-(PPAR-) [22][23][24][25]. These are probably the most relevant mechanisms of action of PEA related to immunopathology.

Metabolism of PEA
7.1. Synthesis. In the body, PEA is synthesized from palmitic acid (C16:0), which is the most common fatty acid in animals and a product of normal fatty acid synthesis. Palmitic acid is also present in many foodstuffs including palm tree oil, meats, cheeses, butter, and dairy products. Synthesis of PEA takes place in membranes of various cell types and involves different steps and partly parallel pathways. The most studied pathway goes via N-palmitoyl-phosphatidyl-ethanolamine, which belongs to the class of N-acylphosphatidylethanolamines (NAPEs). NAPEs in general are present in phospholipid membranes and function as stable precursors and source of their respective NAEs. Palmitic acid is incorporated from the sn-1 position of a donor phospholipid like phosphatidylcholine and transferred to an ethanolamine phospholipid, for example, phosphatidylethanolamine, which is catalysed by a Ca 2+ -dependent N-acyltransferase [26][27][28]. Next, free PEA can be generated by a NAPE-hydrolyzing phospholipase D (NAPE-PLD). However, recent studies also demonstrate the presence of NAPE-PLD-independent multistep pathways to form NAEs from NAPE [28].
An alternative pathway involves formation of NAEs from N-acylated plasmalogen-type ethanolamine phospholipid (N-acyl-plasmenylethanolamine) through both NAPE-PLDdependent and independent pathways [28]. In general, tissue patterns of NAEs are considered to reflect the local availability of their precursor fatty acids in the phospholipid membranes, which are amongst others diet-related [29]. However, in case of PEA, tissue levels hardly seem to be influenced by variation in intake of dietary fatty acids, except in the small intestine where dietary fat results in decreased levels of PEA and other NAEs [27,30]. Several studies indicate that free PEA levels increase during inflammation [27,30,31]. Concentrations of PEA in tissues and plasma have been published in various papers, as recently reviewed by [27,30,31]. In humans, PEA plasma concentrations are subject to considerable variation during the day [32].

Breakdown.
Like with other NEAs, endogenous PEA is produced on demand and acts locally. Tissue levels are tightly regulated through a balance between synthesis and breakdown. The primary degrading enzyme is fatty acid amide hydrolase (FAAH, now also known as FAAH-1), localised on the endoplasmatic reticulum [33]. A second FAAH enzyme, now called FAAH-2, was found in humans, located on cytoplasmic lipid droplets [33,34]. Recently, a third NAE hydrolysing enzyme, N-acyl ethanolaminehydrolyzing acid amidase (NAAA) has been identified [28]. In the cytosol, fatty-acid binding proteins and heat-shock proteins may serve as carriers for PEA to their degrading enzymes [27].

PEA Anti-Influenza Activity: Decrease of Proinflammatory Cytokines
After an infection with the influenza virus, the immune system reacts by an increased production of many cytokinepatterns. One pattern is related to a proinflammatory response and a second one to an antiviral response. Infections with virulent influenza viruses together with an aberrant and excessive cytokine production are linked to increased morbidity and mortality [35]. Increased production of specific inflammatory cytokines, such as the tumor necrosis factor (TNF)-, interleukin-(IL-) 1, IL-6, and IL-10, is characteristic during an influenza infection [36]. More virulent viruses are also associated with rapid and sustained induction of inflammatory cytokines and such an early dysregulation of the host response is seen as contributing to the severity and outcome of the infection [37,38]. The increased production of proinflammatory cytokines, hypercytokinemia, is thus 6 International Journal of Inflammation a clear player in the disease progression and death of patients infected by influenza viruses [39,40]. Recently, it was demonstrated that highly elevated levels of serum IL-6 and IL-10 in A (H1N1) patients may also lead to disease progression [41].
Overactive and nonfunctional hyper induction of proinflammatory cytokines might therefore play a key role in the symptomatology and may lead to increased morbidity and mortality. PEA is widely known for its anti-inflammatory activity and to date more than 60 PubMed indexed papers discuss this property of PEA. Its inhibitory action on TNFalpha secretion is sufficiently documented [42]. But PEA has a much wider modulating effect on interleukins. For instance, recently PEA was shown to significantly attenuate the degree of intestinal injury and inflammation and to inhibit proinflammatory cytokine production (TNF-, IL-1 ), adhesion molecules (ICAM-1, P-selectin) expression, and NF-B expression [43]. PEA also significantly decreases inflammation caused by ischemia-reperfusion injury, a pathological state characterized by a strong enhanced interleukin-cascade [44]. As PEA downmodulates a number of proinflammatory cytokines, this could very well be the reason for the decreased influenza and common cold symptomatology in individuals treated with PEA.

Conclusions and Therapeutic Perspective
Over 350 papers have been referenced in PubMed in the last 50 years describing the physiological properties of PEA and its pharmacological and therapeutical profile. PEA has a broad spectrum of biological targets and target molecules, among which are PPAR-alpha, TRPV1, and orphan receptors such as GPR-55.
This review on the role of PEA as an anti-inflammatory agent and as a therapeutic agent for influenza and the common cold discusses 6 clinical trials in a total of nearly 4000 patients and volunteers where PEA's effectiveness and safety for the treatment in these indications was shown. Furthermore, since the focus on respiratory inflammation and flu between 1971 and 1980, PEA has also been extensively tested in a great variety of animal models for a number of other indications, such as central and peripheral neuropathic pain, pain in osteoarthritis, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, irritable bowel disease, interstitial cystitis, and other visceral pain states. Consistently, the effective dose range has been between 10 and 30 mg PEA/kg bodyweight [45,46]. Since the work of Levi-Montalcini in the 90s of the twentieth century, results of around 40 clinical trials in chronic pain have been reported. However, the majority of these results were reported in Italian and Spanish medical journals [37]. Since 2008, an increasing number of clinical data have been reported in English literature and results support its use in indications such as sciatic pain and related neuropathic pain disorders. As PEA clearly plays a fundamental role as a protective and restorative modulating lipid precursor, its clinical role is currently further evaluated in a variety of disorders such as inflammatory bowel disorder, central neuropathic pain in spinal cord disorders, various eye disorders such as glaucoma and degenerative retina disorders, multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease.
Given the results of 6 clinical trials in flu and the common cold, seen in the context of the serious criticism on the efficacy and safety of oseltamivir and zanamivir, PEA should be reconsidered by clinicians as a new treatment modality for the flu and respiratory infections due to its documented efficacy and more importantly its very benign side effect profile. Furthermore, oseltamivir and zanamivir are known to induce resistance; PEA has a very low likelihood of inducing resistance due to its mechanism of action. Finally, the ease of application of PEA offers the possibility to have a quick therapeutic answer ready in case of a flu epidemic, especially in cases of a mismatch between circulating strains and the recommendations from WHO.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
PIK3CA hypomethylation plays a key role in activation of the PI3K/AKT pathway in esophageal cancer in Chinese patients.

AIM
To investigate the role of PIK3CA oncogene in tumorigenesis and development of esophageal cancer in Chinese patients at the levels of genetic mutation and epigenetics.

METHODS
Seventy six esophageal tumor samples and corresponding adjacent normal tissues were collected, and the genomic DNA was extracted. Mutations in the 9th and 20th exons of PIK3CA gene were detected using conventional sequencing. PIK3CA methylation rates in two selected CpG islands (CpG island 1 and 2) were detected using sub-bisulfate modified sequencing. P110α and pAKT expression levels were detected with Western blotting.

RESULTS
In PIK3CA gene of the tumor tissues, G1633C (E545Q) mutation was detected in the 9th exon with a rate of 3.95% (3/76), whereas mutation was not found in the 20th exon. Nor mutation did occur in PIK3CA gene of the adjacent normal tissues. The methylation rate of the CpG island 1 had no significant difference between the tumor and adjacent tissues (0.77%±0.009% vs 0.89%±0.008%), but the methylation rate of the CpG island 2 in the esophageal tumors was significantly lower than that in the adjacent tissues (6.00%±2.80% vs 10.45%±5.51%). Furthermore, the rate of methylation of the CpG island 2 in TNM stage III and IV esophageal cancer (3.84%±2.08%) was significantly lower than in stage I (8.52%±2.55%) and stage II (6.42%±2.36%). PIK3CA gene hypomethylation in esophageal cancer was significantly correlated with high expression of p110α.

CONCLUSION
PIK3CA gene hypomethylation plays a key role in the tumorigenesis and development of esophageal cancer in Chinese patients, while the mutations of PIK3CA gene have little effect on the development of esophageal cancer.

Introduction
In China, esophageal cancer is one of the three most common tumors of the gastrointestinal tract, together with gastric and colorectal cancer, and it has the fourth highest mortality rate of all malignancies. The mortality rate due to esophageal cancer was 15.04/100 000 in [2004][2005], with rates of 20.46/100 000 and 9.38/100 000 in males and females, respectively [1] . The highest incidence of esophageal cancer occurs in Henan Province, China, and is 81.78/100 000 [2] . It remains a challenge to give effective therapy for esophageal patients due to the lack of targeted therapeutic drugs. Therefore, there is an urgent need to investigate the mechanism of the development of esophageal cancer.
PIK3CA is one of the most common oncogenes in human cancers [3] . P110α, the product encoded by the PIK3CA gene, is a catalytic subunit of PI3K. Phosphate kinase (PIK) is a lipid kinase that phosphorylates phosphoinositide and can participate in signal transduction. Based on the phosphorylation sites, the PIKs can be divided into three families: phosphoinositide 3-kinase (PI3Ks), phosphoinositide 4-kinase (PI4Ks), and phosphoinositide 5-kinase (PI5Ks). The PI3Ks are further divided into three classes (I, II, and III) according to subunit structure, regulation and substrate selectivity. The Class I PI3Ks contains the IA and IB subgroups. Class IA PI3K is composed of three catalytic subunits: p110α, p110β, and p110δ, and five different regulatory domains: p85α, p85β, p85γ, p50α, and p55α. These PI3Ks are activated by the cell surface receptor-tyrosine kinase. Class I PI3Ks catalyze the formation of phosphatidylinositol-3-phosphate (PIP3). PIP3 combines with the serine/threonine kinases AKT1, AKT2, and AKT3, targets the cell membrane and is then activated by 3-phosphoinositide-dependent protein kinase 1 (PDK1). After activation, AKT displays a wide range of biological effects through its series of target proteins, such as mTOR, BAD, caspase 9, tuberin, GSK3β, MDM2, p21, FKHR-L1 [4][5][6][7][8][9][10][11] , and several forkhead transcription factors. These effects are related to the regulation of cell proliferation and cell survival, and the control of cell movement. PIK3CA overexpression, caused by changes in the PIK3CA gene, enhances PI3K catalytic activity, while the intracellular PI3K/AKT pathway maintains an abnormal activation status and is responsible for malignant transformation.
Mutations of PIK3CA exist in approximately 30% of human solid tumors. The tumors that most frequently display the PIK3CA mutation include endometrial, breast, colon, and skin cancers [3] . According to the literature, PIK3CA mutation rates in esophageal cancer are lower in Japanese and Australian patients [12,13] . We found no experimental data on PIK3CA methylation in the published literature. We aimed to investigate the relationship between PIK3CA and esophageal cancer with regard to mutation and methylation.

Materials and methods
Patients and pathologic specimens All 76 cases of esophageal cancer underwent surgery in the Department of Cardiothoracic Surgery of the First Affiliated Hospital of Soochow University between May 2010 and May 2011. The pathologic specimens were processed within 10 min of surgical resection. All adjacent tissues were removed from surgical sites at least 5 cm from the tumor margins. The specimens were immediately frozen in liquid nitrogen and stored at -80 °C. The patients had not received any preoperative therapy, including chemotherapy, radiotherapy and biotherapy. All patients underwent radical resection and were confirmed by pathological diagnosis. Two patients had adenocarcinoma, and the rest had squamous cell carcinomas. This study was approved by our institutional ethics committee.
Genomic DNA extraction Genomic DNA was extracted from 76 frozen fresh tissues using a UNIQ-10 Genomic DNA Purification Kit (Shanghai Sangon Biotech Co, Ltd, Shanghai, China) according to the manufacturer's instructions.
We conducted bisulfite sequencing PCR (BSP). Bisulfite DNA conversion was performed according to the manufacturer's instructions included with the EpiTect Bisulfite Kit (Qiagen, Duesseldorf, Germany). A 5 µg sample of purified DNA was brought to a total volume of 140 µL. The bisulfite reactions were mixed thoroughly, and the tubes were placed at room temperature. Bisulfite DNA conversion was performed with a thermal cycler as follows: denaturation for 5 min at 95 °C, incubation for 25 min at 60 °C; denaturation for 5 min at 95 °C, incubation for 85 min at 60 °C; denaturation for 5 min at 95 °C, incubation for 85 min at 60 °C; hold at 20 °C. After the bisulfite conversion was complete, purified bisulfite-treated DNA was used as a template for PCR using HotStar Taq DNA polymerase (Qiagen, Duesseldorf, Germany) with specific primers. The PCR products were separated on 1.5% agarose gels and purified using the AxyPrep Gel Extraction Kit (Axygen, Tewksbury, MA, USA). The purified DNA fragments were ligated into the pGEM ® -T vector systems (Promega, Madison, WI, USA) and transformed into TOP10 competent cells (Invitrogen, Grand Island, NY, USA) for further replication. Correct recombinants were screened using the IPTG/ x-Gal system, and the plasmids were extracted and sequenced using an ABI 3730 XL.
The amplification products were sequenced by Shanghai HaoYu Biotechnology Co, Ltd (Shanghai, China). We used chrome AC/DC to observe the ABI peak figure files and to determine whether these peaks represented singlebase mutations or base deletions. Statistical  We used Quantity One software (Bio-Rad, Hercules, CA, USA)to test each sample's gray scale values of β-actin and target protein bands. According to the data presented above, we calculated the relative content of the target protein and compared the samples to determine the actual change in the level of the target protein between samples. We found that the gray scale values of the target protein were 50% higher in esophageal cancer than in the adjacent tissues. There was no difference in expression, with positive expression numbers of pAKT and p110α, respectively.

Statistical analysis
All data were categorized using Excel 2003. Statistical analysis was performed using SPSS 18.0 statistical software. All experimental data are expressed as the mean plus or minus the standard deviation. Comparisons of variable data were performed with the chi-squared or Fisher's exact test (double sided), Spearman correlation analysis, as well as the Student's t-test and ANOVA for single comparisons. A P-value of less than 0.05 was considered statistically significant.

PIK3CA mutation in esophageal cancer patients
We detected a G1633C (E545Q) mutation in the 9th exon of PIK3CA in 3.95% (3/76) of the esophageal squamous cell carcinomas. No mutations were found in the 20th exon of PIK3CA in the tumor tissues or the adjacent tissues. The PIK3CA mutation is a point mutation [12] that occurs during the middle to late stage of esophageal squamous carcinoma (Figure 1 and  2).

PIK3CA methylation
We used 32 samples of esophageal tumor tissue and the corresponding adjacent tissues for the methylation determination.
MethPrimer software (The Li Lab, San Francisco, CA, USA) was used to predict the human PIK3CA gene promoter regions, and we selected two CpG islands rich in CG (CpG island 1: -800 bp to -400 bp, CpG island 2: -250 bp to 50 bp). Five PCR clones from each specimen were selected for sequencing (Figure 3 and 4).

The relationship between PIK3CA hypomethylation and demo graphic features in esophageal cancer
We analyzed the relationship between PIK3CA methylation and the demographic characteristics of patients with esophageal cancer. There was a significant difference in the average methylation rate at Stage III and IV (3.84%±2.08%) compared with Stage I (8.52%±2.55%) and II (6.42%±2.36%) (P I-III =0.001, P II-III =0.01). There were no other statistically significant differences (Table 1).  Figure 6). We demonstrated that p110α expression was significantly higher in the tumor tissues versus the adjacent tissues  Table 2).

Discussion
PIK3CA mutation in esophageal cancer occurs in the middle and advanced stages PIK3CA mutations occur in approximately 30% of human   [3] . The PIK3CA mutation rates vary from 4%-52% in different types of cancer [17][18][19][20][21][22][23][24][25][26][27] . In colon cancer, the PIK3CA muta-  tion rates are over 30% and are the highest among all gastrointestinal tumors [28,29] , followed by gastric cancer, with mutation rates that vary from 4%-25% [30,31] . In esophageal cancer, the PIK3CA mutation rate is as high as 21% [32] . Of these mutations, 80% are clustered in two previously reported "hotspots" or high-risk regions of the 9th and 20th exons, which correspond to the accessory (helical) and catalytic domains of PIK3CA [3] . Non-random hypermutation plays a significant role in tumor development. The main types of mutation are E545K (G1633A) and E545Q (G1633C) [33] , E542K, and E545D [12] , and A1634C [13] in the 9th exon and H1047R in the 20th exon [34] . Our study focused on these two exons, and we found that all of the PIK3CA mutations in our samples were G1633C. This point mutation occurred at a rate that was lower than reported in other studies. We found no substitutions, truncations, or frameshift mutations [12,35] . Thus far, the role of PIK3CA mutations in esophageal carcinogenesis (early stage) or the promotion of malignancy (advanced stages) has not been established. Some investi-    gators speculate that PIK3CA mutations may play a role in early esophageal tumors and subsequent carcinogenesis [12] .
The role of PIK3CA in gastric cancer has been established; the lack of associated symptoms or pathology suggests that PIK3CA mutations may occur early in cancer development [36] .
Yardena et al found that PIK3CA mutations in early colon cancer occurred at a much lower rate than in middle-late stage cancer; thus, a relationship between PIK3CA mutations and carcinogenesis was proposed [35] . Other investigators have demonstrated that PIK3CA mutations occur almost exclusively in invasive tumors, with upstream gene mutations often occurring in early and late tumors, suggesting that PIK3CA mutation is a late event [37] . We demonstrated that esophageal PIK3CA mutations only occurred in the later TNM stages (IIIa and IIIb). While there is abundant evidence that PIK3CA mutations promote tumorigenesis by activation of the PI3K/ AKT pathway in other cancers [38,39] , we did not find an association between p110α and pAKT expression in the PI3K/AKT pathway. Therefore, the PIK3CA mutation may occur late in esophageal cancer. We found no evidence to support that these mutations promote malignant degeneration. Additional studies are required to determine the role of PIK3CA in the development of esophageal cancer, including investigations with a broader range of detection of mutations and studies of additional esophageal cancer cases.
PIK3CA hypomethylation is closely related to the activation of the PI3K/AKT pathway and may be a key mechanism in esophageal carcinogenesis DNA methylation is an important mode of epigenetic modification, and it occurs in a variety of context throughout the mammalian genome. CpG occurs in either a dispersed (scattered) or a highly concentrated ("CpG island") pattern. CpG islands occur in up to 1% of genomic DNA, and they are present at the 5' ends of the regulatory regions in all housekeeping genes. CpG islands often cover the promoter region and are found within the first exon. Hypomethylation is another abnormal form of DNA methylation in cancer. Hypermethylation of a suppressor gene and hypomethylation of oncogenes may coexists in cancer, which can affect the stability of the genome and the normal expression of gene involved in cell proliferation and differentiation. These aberrations can cause cells to lose control of their normal processes, promoting the formation and development of tumors. In 1983, Feinberg and Vogelstein found that the growth hormone genes, α-globin and β-globin, are highly methylated in normal tissues but not in cancer tissues [40] . Universal hypomethylation of the entire genome and hypomethylation of specific sites in the oncogene promoter region increases cancer risk [41] . The lower degree of methylation in proto-oncogenes is significantly related to invasiveness [42,43] . There are no reports about PIK3CA methylation in esophageal cancer to date. The PIK3CA oncogene should be hypomethylated in esophageal cancer and hypermethylated in adjacent or normal tissues. We found that the PIK3CA methylation rate in esophageal cancer was significantly lower than in the adjacent tissues, correlating with higher TNM stag-ing.
In addition, our data show that PIK3CA methylation negatively correlates with p110α expression, but not pAKT expression. That is to say, the status of PIK3CA hypomethylation in esophageal cancer regulates its coding expression. Meanwhile, other inhibitory factors may be reduced, resulting in stimulation of the expression of p110α. The reason for the absence of a correlation with the downstream target molecule-pAKT, based on our understanding of the PI3K/AKT signaling pathway, we proposed that pAKT is the terminal target molecule of multiple signaling pathways, and its expression level is influenced by many factors. Therefore, PIK3CA hypomethylation is not the decisive factor determining the high level of pAKT expression.
Our previous experiments [44] have demonstrated that PI3K/ AKT activation is closely related to the progression of esophageal cancer. Thus, we believe that PIK3CA hypomethylation is significantly related to the development of esophageal cancer. PIK3CA hypomethylation may be part of the activation mechanism in the PI3K/AKT pathway.
We only selected two CpG islands based on the CpGplot program. The full effects of PIK3CA methylation may not be completely apparent due to missing methylation sites. In our future experiments, we will obtain additional samples and examine other CpG islands. Hypermethylation of PTEN, which is related to the PI3K/AKT pathway, may be carcinogenic in esophageal cancer. Whether there is a link between PTEN hypermethylation and PIK3CA hypomethylation remains unknown and requires further investigation.


extfieldsofstudy: Biology Medicine

text:
Structure-Function-Immunogenicity Studies of PfEMP1 Domain DBL2βPF11_0521, a Malaria Parasite Ligand for ICAM-1

Plasmodium falciparum virulence has been ascribed to its ability to sequester in deep vascular beds, mediated by the variant surface antigen family PfEMP1 binding endothelial receptors like ICAM-1. We previously observed that naturally-acquired antibodies that block a PfEMP1 domain, DBL2β of PF11_0521 allele, from binding to the human ICAM1 receptor, reduce the risk of malaria hospitalization in children. Here, we find that DBL2βPF11_0521 binds ICAM-1 in the low nM range and relate the structure of this domain with its function and immunogenicity. We demonstrate that the interaction with ICAM-1 is not impaired by point mutations in the N-terminal subdomain or in the flexible Loop 4 of DBL2βPF11_0521, although both substructures were previously implicated in binding ICAM-1. These data will help to refine the existing model of DBLβ::ICAM-1 interactions. Antibodies raised against full-length DBL2βPF11_0521, but not truncated forms lacking the N terminal fragment, block its interaction with ICAM-1. Our data suggest that full length domain is optimal for displaying functional epitopes and has a broad surface of interaction with ICAM-1 that is not disrupted by individual amino acid substitutions at putative key residues. This information might be important for the future design of anti-malarial vaccines based on PfEMP1 antigens.

Introduction
Cytoadherence plays an important role in the lifecycle and virulence of P. falciparum, the deadliest of human malaria parasites. The sequestered mass of adherent parasites leads to vascular occlusion that strongly correlates with disease severity [1] and inflammation in different organs. The main parasite ligand for infected erythrocyte (IE) adhesion is a family (,60 members) of PfEMP1 proteins [2][3][4] that appear on the surface of IE in specialized structures called ''knobs'' [5]. Cytoadhesion of IE is a cause or feature of cerebral malaria (CM) [6][7][8] and of pregnancy malaria (PM) [9].
Each PfEMP1 protein consists of single intracellular and transmembrane domains, and several extracellular domains (2 to 7 domains, ,30-45 kDa per domain) heavily cross-linked by disulfide bonds. All PfEMP1 domains have been classified into several sub-classes: Duffy binding like (DBL) a, b (previously called bC2, a combination of the b and C2 domains), c, d, f, e, and X; and cysteine-rich interdomain regions (CIDR) a, b, and c [4,10,11]. DBL domains all have a similar scaffold [12][13][14][15]; the C-terminal part of the CIDR scaffold is similar to the C-terminal part of the DBL domain [16]. These domains are responsible for adhesion to various host receptors (reviewed in [17]). The host receptor binding specificity and combination of individual domains in the expressed PfEMP1 proteins may determine final IE adhesion specificity, strength of binding, and possibly, different pathologies related to malaria syndromes [18].
Various refinements of domain classification and of domain boundaries resulted from recent analyses of PfEMP1 sequences from 7 parasite genomes [19]. For example, in the previous terminology DBLb and C2 were considered as separate domains that are found always together as a DBLbC2 combination. However, domain C2 is essentially a part of the same b domain [20,21]. Therefore, DBLb is a more correct term for this class of domains [19] which we will use henceforth.
Though its role remains inconclusive, ICAM-1 has been proposed as the key host adhesion receptor for iRBC causing CM [7,8]. In support of this, parasites isolated from patients with CM bind at higher levels to ICAM-1 [22]. Domains of the DBLb class have been implicated as ligands for ICAM-1 [10], although most DBLb domains do not bind this host receptor. For example, only 6/24 DBLb domains from the IT4 genome bound to ICAM1 in vitro [20]. In our previous work, only 1/18 DBLb domain from the 3D7 genome, specifically the DBL2b domain of PF11_0521 allele, showed strong avidity binding to ICAM-1 [21]. We also recently observed that antibodies to DBL2b PF11_0521 predict reduced risk of hospitalization for malaria among Tanzanian children [23]. Therefore, understanding the molecular details of the interaction between ICAM-1 receptor and the DBL2b domains, and the structural features leading to a functional immune response against these domains might guide the design of anti-severe malaria vaccines and drugs.
Interactions between ICAM-1 and the DBL2b domain were previously modeled in silico, based on several lines of evidence, such as the key role of a single amino acid residue in loop 4 of the DBL2b domain [24]. We have subsequently shown [21] that DBL2b domains are about 70 amino acid residues longer than previously assumed [10], and that this extra-N-terminal fragment (not included in the in silico model) is important for functional activity [21]. Here, we use site-specific mutagenesis of residues in putative key sub-structures of the DBL2b PF11_0521 domain to assess their roles in binding ICAM-1. We also examined the functional anti-adhesion activity of antibodies raised against truncated and full-length variants of DBL2b PF11_0521 to better understand the requirements for PfEMP1 domain-based vaccines that might prevent iRBC adhesion and thus severe malaria.

Ethics statement
Animal ethics adhered to specific national and international guidelines. Animal use protocols ''Antibodies Inc. Protocol (Offsite)'' and ''Liver Stage Vaccines'' meet the standards of the Guide for the Care and Use of Laboratory Animals (by National Academy of Sciences) and applicable Seattle Biomedical Research Institute (Seattle BioMed) policies and procedures. Seattle BioMed has an Animal Welfare Assurance (A36640-01) on file with the NIH Office of Laboratory Animal Welfare. Protocols #AO-06-ABP and #AO-02 have been approved by Seattle BioMed IACUC committee. All animals used in the experiments were observed on a daily routine for presence or absence of distress and/or signs of illness, timely veterinary care was provided as needed. Euthanasia method via exsanguination by cardiac puncture under ketamine/ xylazine anesthesia was used.

DBL2b PF11_0521 constructs
Cloning of the full-length DBL2b PF11_0521 domain and its expression in COS-7 cells as a surface-expressed molecule was described in [21]. Figure 1 schematically shows full-length DBL2b PF11_0521 domain expressed in COS-7 cells and several domain structural features, as well as constructs (expressed in E. coli) used in this work. The nucleotide and corresponding protein sequences for this domain, the various truncation and pointmutation constructs, as well as names of relevant cloning vectors are all shown in Figure S1. The following amino acid (AA) substitutions were made using Quick Change mutagenesis [25] with Pfu turbo polymerase (Stratagene) in the full-length domain that was cloned into pHisAdEx vector [21] for expression in COS-7 cells: A25K; R23A+A25K; A347L; A347H; A347Y.
Full-length DBL2b domain (AA 1 through 520 corresponding to AA 728-1247 in the sequence of full-length PF11_0521 PfEMP1 protein, starting from its initiator methionine) and its truncated fragments AA 91-391 (''short''), AA 267-391 (''3helix''), and the N-terminal sub-domain (N-terminal fragment, NTF, AA 1-69) were cloned into the pET28b vector, expressed in E. coli, purified by Ni-column followed by Reverse Phase HPLC and refolded as described earlier [26]. These proteins were assessed for binding to ICAM-1 and used for preparation of antibodies.
We expressed HisAdEx [21] without additional insert in COS-7, and AMA-1 [27] in E. coli using pET28b with subsequent purification and re-folding by the same methods described above for full-length DBL2b PF11_0521 domain.

DBL2b PF11_0521 and ICAM-1 binding and bindinginhibition assays
We performed binding and binding-inhibition studies as previously described, using BioPlex (BioRad) approach, COS-7 expressed proteins, and ICAM-1-Fc chimera (R&D Systems) [21]. All measurements were made in duplicate. In each experiment, the amount of recombinant domain immobilized on the beads was measured by reactivity with antibodies to full length E.coliexpressed/refolded domain (see below), and this amount then used to normalize ICAM-1-binding levels [27]. E. coli-expressed and purified domains were immobilized on carboxy-modified polystyrene beads (BioRad) using the manufacturer-supplied protocol and assayed in the same fashion as bead-immobilized COS-7-expressed proteins. COS7-expressed GFP-fused proteins were immobilized on the beads through capture by anti-GFP antibodies previously immobilized by chemical cross-linking [21] and E. coli-expressed domains through direct random crosslinking. For comparison of specific binding activity of E. coli and COS-7 expressed proteins, the amount of protein bound to the beads was measured by reactivity with anti-full length DBL2b PF11_0521 domain sera (rat sera to E.coli-expressed domain) as described below.

Preparation of antibodies against domain variants
Purified and refolded E. coli-expressed domain variants were used for preparation of antibodies in rats at Antibodies, Inc. (Davis, CA) using their standard schedule protocol and 10 mg of antigen per injection, and in mice at Seattle BioMed as described earlier [26]. DNA immunization using full-length domain cloned into pHisAdEx was performed at the Seattle BioMed animal facility as follows: female Balb/c mice aged 6-12 weeks received 4 immunizations (,50 mg DNA) at 3-week intervals intramuscularly. Terminal bleeds were collected 10-14 days after the final immunization. Animal work was described in protocols listed in Ethical Statement above and has been approved by IACUC at Seattle BioMed.

Reactivity of anti-domain antibodies with various DBLb PF11_0521 domain constructs
A range of dilutions of anti-sera raised against various domain constructs was incubated with different bead-immobilized domain constructs for 1 hour at room temperature (RT). Pooled preimmune sera and anti-sera from animals raised against control proteins (HisAdEx or AMA1) were used as negative controls. We used pooled sera from all animals immunized with the same antigen. Beads were washed 3 times with phosphate buffered saline (PBS)-0.05% Tween-20 (PBST), incubated with goat antispecies IgG coupled to PE (Jackson Immunoresearch, dilutions: 1:5000 anti-rat, 1:1000 or 1:500 anti-mouse) for 1 hour at RT, washed 3 times in PBST and 1 time with PBS, and finally resuspended in 125 ml of PBS. Signals were measured on BioPlex 200 (BioRad) as described in our publications [21,23].

Determination of avidity constant for binding of DBL2b PF11_0521 and ICAM1
We used full-length DBL2b domain expressed in both systems (COS-7 and E. coli) and immobilized on BioPlex beads (as described above) for these measurements. E. coli-expressed protein was refolded as described above. Initial velocity of binding was measured at various concentrations (0.3, 1, and 3 ug/ml) of ICAM-1-Fc. At these concentrations and incubation times (5 to 35 min), the level of ICAM-1 binding to bead-immobilized domain was less than 10% of the maximum. The incubations and measurements were performed in a homogenous system; ICAM-1-Fc was pre-incubated for 1 hour with an excess of donkey anti-human IgG coupled to phycoerythrin (PE) detection molecules, and then added to beads with immobilized full-length DBL2b domain. Final concentrations of ICAM-1 in these mixtures are shown above and the final dilution of anti-human IgG-PE was 1:250. After incubation for the indicated periods of time, beads were subjected to signal measurements using a Bio-Plex 200 machine (BioRad) without any washing steps. The actual time (in seconds) between mixing ICAM-1-Fc and donkey antihuman IgG-PE with beads and signal measurement events on the machine was used for kinetics calculations. As active ICAM-1-Fc molecules are organized as dimers, we used its molecular mass equal to ,200 kDa (R&D Systems) for calculations. We used the Michaelis-Menten equation and Lineweaver-Burk plots [28] for the calculation of the equilibrium dissociation constant K D . Linear regression lines and 95% confidence intervals (CI) were calculated using GraphPad Prizm software.
Visualization of DBL2b::ICAM-1 interacting molecules and in silico amino acid substitution analyses were performed using functions of Deep View Swiss-PDB viewer and Bertonati and Tramontano model [24].

Results and Discussion
A model of DBL2b::ICAM-1 interactions suggested by Bertonati and Tramontano [24] was obtained by computational docking of the X-ray resolved 3-D structure of ICAM-1 N-terminal domain and the predicted 3-D structure of PfEMP1 DBL2b domain, in turn modeled using the available X-ray resolved 3-D structure of the homologous F1 domain in EBA 175 malarial protein [13,30]. This model explains or is in agreement with several experimental observations regarding PfEMP1 domain::ICAM-1 interactions (discussed in [24]) and may serve as a basis for design of various biochemical experiments for further characterization of DBL2bI-CAM-1 domain interactions.
However, the Bertonati-Tramontano model has limitations, and several observations cannot be explained by this model. For example, the Y-motif in the C2 part of the domain (Figure 1 and Figure S2A) was shown to be critical for ICAM-1 binding [31], and chimeric constructs indicate interdependence between the C2 and the upstream components of the domain [31]. Despite these experimental data, the Y motif of C2 is distant from the ICAM-1 interaction site in the model of Bertonati and Tramontano, and does not make contacts with ICAM-1, nor substantial contacts with the rest of the DBLb domain ( Figure S2A). In addition, a completely conserved Tyr-Ala-Phe (YAF) motif located in the C2 part of the DBLb domain ( Figure 1) is not incorporated in this model. The importance of the C-terminal third of the DBLb domain from another PfEMP1 protein, PFD1235w, for ICAM-1 binding was confirmed recently [32] but the implicated part (,180 AA) includes sequences that are involved in the interactions according to the model as well as those that are distant.
Different parasite strains [21,24,31] consistently have A, L, or H residues in position 286 within flexible loop 4 (AA number according to [24], in our full-length domain this is number 347) of ICAM-1 binding parasites, while non-binding strains contain E, K, Q, W, S, Y as well as A, L, and H. E and K are the most common residues in this position. The Bertonati-Tramontano model predicts that several residues including AA286 in the DBLbdomain make close contact with ICAM-1 ( Figure S2). However, in silico substitutions of amino acid residue 286 in the model [24] that we performed using Deep View Swiss-PDB viewer indicate that even those residues that are present in Loop 4 in ICAM-1 non-binding strains (including E and K) are sterically compatible with the modeled interactions, and may be energetically advantageous (Figure S2 B and C). Therefore, we tested in vitro the effect of substitutions of amino acid residue 286 on DBL2b::ICAM-1 interactions.
Finally, we have shown [21] that the previously defined DBLb domain [10] used for modeling [24] was missing an additional Nterminal subdomain. This has subsequently been confirmed in analyses of seven P. falciparum genomes [19]. Moreover, for the 3D7 DBL2b PF11_0521 domain, which strongly and specifically binds ICAM-1, we reported that the N-terminal subdomain contributes to ICAM-1 binding activity [21]. Therefore, we also made point mutations in a conserved alpha-helix ( Figure S3) predicted by consensus secondary-structure analysis [29] to be a prominent structural element in this N-terminal subdomain [21]. As we wanted to preserve the structure of this alpha-helix but disturb possible interactions, we made substitutions that significantly changed the physical-chemical nature of the amino acid residues but did not affect the formation of this a-helix. Secondary structure analysis of our point mutation sequence variants according to Deleage et al. [29] predicts preservation of this ahelix.
As expected for two proteins with substantial surface area contacts, none of the single point mutations in the DBLb domain diminished binding interactions with ICAM-1 (Figure 2), presumably because they did not disrupt overall folding of the protein.
Preservation of overall folding would be expected for the substitutions made in flexible loops. Similar results were obtained in previous work in which Alanine replacements of at least 3 residues (different from those reported here) were required to reduce binding interactions [20]. In our mutation experiments, the substitution of a natural Ala286 residue in Loop 4 (number 347 in our full-length construct) to any of the tested residues did not reduce interactions. Moreover, substitution with Tyr, the residue that occurs only in ICAM-1 non-binding variants, yielded an even stronger interaction with ICAM-1, in agreement with our in silico modeling of Tyr substitution (above) that predicts an additional intra-molecular hydrogen bond (between Tyr 186 and Arg113 of the domain) that may stabilize the complex ( Figure S2 B and C). However, this agreement with the Bertonati and Tramontano model does not explain the observation that only A, L, and rarely H residues appear at this position in ICAM-1 binding DBLb domain variants. More DBLb domain variants should be tested in order to strengthen or refute the association of A, L and H residues at this position, with binding to ICAM-1.
Similar to the Loop 4 findings, substitutions in the N-terminal subdomain a-helix did not affect ICAM-1 binding (Figure 2). Thus, these amino acid residues are not crucial for interactions with ICAM-1. These data further support the notion that the two molecules have substantial surface interactions that are not affected significantly by point mutations that preserve overall structure.
We have expressed four structural variants of DBL2b PF11_0521 domain (Figures 1 and S1) in E. coli, then purified and refolded (at least made soluble) these: the full length domain (sequence as described in [21]); a truncated construct designed according to the minimal binding fragment (MBF) [31] and called ''short''; a construct called ''3-helix'' that comprises the three helices involved in forming Loop 4 ( Figure S2), which interacts with ICAM-1 according to the model [24]; and N-terminal subdomain (also called here as N-terminal fragment, NTF). Of note, the proposed MBF does not bind ICAM-1 [31]: although a series of sequential DBLbdomain truncations performed independently at the N-and C-termini suggested this fragment to be the MBF, it is not functional as a single construct.
In accordance with our previous results using COS-expressed truncated domains [21], the ''short'' and ''3-helix'' DBL2b PF11_0521 domain variants did not bind ICAM-1 (data not shown), most likely due to incorrect folding of these truncated domain fragments (see below). Refolded full-length recombinant domain strongly bound the ICAM-1 receptor (Figure 3), though the specific activity (ICAM-1 binding per amount of domain immobilized on the bead as measured using antibody to E. coliexpressed full length domain, described below) was ,3-fold lower than that seen with COS-7-expressed protein. This indicates that only a fraction of the E. coli preparation (,33%) is refolded correctly to the active conformation or is sufficiently stable for binding on the beads. Random immobilization (of the E. coliexpressed protein) may inactivate some number of molecules by their unfavorable cross-linking on the surface of the beads. This may contribute to the inactive pool of bead-immobilized domain molecules and indicates that at least 33% of the refolded soluble preparation was active before cross-linking. Directed immobilization (of the COS-expressed domain) in this respect is preferable over the random immobilization.
Binding between the DBL2b PF11_0521 domain and ICAM-1 receptor is strong, with a calculated equilibrium dissociation constant (K D ) in the low nM range (2.3-2.6 nM; 95% CI = 1.3-4.4 nM) ( Figure S4). The high avidity receptor binding is in accordance with the notion that parasite cytoadherence may continue for many hours after killing parasites with conventional drugs, contributing to pathophysiology of severe disease [33], and with the presumptive role for ICAM-1 receptor in severe malaria [10,22]. The strong avidity constant for ICAM-1 binding also supports previous results on the important role for ICAM-1 receptor in tight binding of infected erythrocytes to endothelial cells under flow [34]. High avidity binding can also explain why naturally acquired antibodies fail to reverse this interaction [21,35] even though they efficiently block it [21]. Most adults but not children living in malaria endemic regions have acquired antibodies that block binding to ICAM1 [21,23], and these might protect against severe malaria by limiting new parasites from sequestering, even though binding is difficult to reverse once adhesion has occurred.
We have previously demonstrated that antibodies against DBL2b PF11_0521 domain are common in malaria-immune adults [21], appear early in life, and are associated with protection against severe disease in young children [23]. Therefore, we were interested to determine whether antibodies raised against the recombinant DBL2b PF11_0521 domain would affect binding of this domain to ICAM-1, and as well to determine the structure and size of the domain needed to re-create functional epitopes. All four E. coli-expressed DBL2b PF11_0521 domain constructs (full-length, ''short'', ''3-helix'', and NTF) were used for immunizations of rodents, as was plasmid DNA pHisAdEx-DBL2b PF11_0521 containing full length domain. This latter construct was previously used for production of functionally active protein in COS-7 cells Characterization of ICAM-1 Binding PfEMP1 Domain PLOS ONE | www.plosone.org [21] and is the base construct for mutagenesis and ICAM-1 binding experiments in this work.
Mouse anti-full-length domain antibodies prepared by DNA immunization recognized both E. coli and COS-7 expressed fulllength domains ( Figure 4A), with similar endpoint titers down to 1:64,000. The level of reactivity was higher for the beads displaying the E. coli-expressed domain because more protein bound to these beads, compared to beads displaying COS-7 expressed protein (as was found using antibodies raised against E. coli-expressed domain, see below). Antisera collected after DNA immunization failed to react to the ''short'' E. coli expressed truncated domain, which represents a substantial portion of the full-length protein but, as we suggested, is mis-or un-folded. We conclude that intramuscular DNA immunization yielded correctly folded domain on the surface of the host cells and induced antibodies that predominantly recognize surface-exposed structural epitopes of this correctly folded form, whereas antibodies raised against E. coli-expressed protein (see below) recognize epitopes of both folded and unfolded protein and might therefore reflect total protein in a preparation rather than solely the binding form of protein. Because antibodies raised by DNA immunization recognize only the folded form of the domain, they cannot be used to determine the amount of total protein immobilized on the beads.
Rat antisera against E. coli expressed/refolded full-length domain recognized both E. coli expressed/refolded full length domain and COS-7 expressed domain ( Figure 4B), with titers down to 1:4,096,000. These antisera also strongly reacted with inactive ''short'' mis/unfolded domain ( Figure 4B), presumably because a fraction of the E. coli product used for immunization included mis-or un-folded protein. Using these antisera, we quantified the amount of protein on the BioPlex beads, and then estimated the specific (normalized) binding activity of mutant variants, and compared specific ICAM-1 binding activity of E. coli expressed/refolded and COS-7 expressed protein (above). Rat anti-''short'' antibodies strongly and comparably recognized full-length and ''short'' E. coli expressed domains but recognition of COS-7 expressed full-length domain was significantly weaker (,12 times less reactivity normalized to the amount of protein on beads) ( Figure 4C, top), again indicating that the majority of the COS-7 expressed protein is folded and that cryptic or linear epitopes, the main targets of ''short'' domain antigen, are not accessible. However, the recognition is clearly detectable using a lower dilution of secondary antibodies (exemplified in Figure 4C, bottom, using antibodies raised in mice), indicating that these antibodies either recognize some small number of epitopes (most likely linear) in the full-length domain, or the recognition is of low affinity, or the COS-7 expressed preparation also contains some small amount of mis-or un-folded protein (or a combination of all three). These antibodies did not inhibit binding of the COSexpressed domain to ICAM-1 ( Figure 5A). Similar behavior was observed for antibodies prepared in rats and mice. Mouse anti-''3helix'' antibodies behaved similarly and did not inhibit ICAM-1 binding ( Figure 5A) but had slightly higher reactivity than anti-''short'' antibodies ( Figure 4C, bottom). Finally, rat anti-NTF antibodies behaved the same way as antibodies to both Nterminally truncated fragments (''short'' and ''3-helix''): they strongly recognized E. coli-expressed NTF and full-length domain but poorly recognized COS-expressed full-length domain and did not inhibit ICAM-1 binding (data not shown).
Antibodies raised against full-length domain (using DNA immunization or immunization with refolded E. coli expressed protein) were functionally active as they can completely inhibit ICAM-1 binding to COS-expressed DBL2b domain ( Figure 5B). The titer of the antibodies obtained by DNA immunization was significantly lower than that obtained by protein immunization; the inhibitory activity was proportionally lower.
In summary, our results indicate that the prevailing model of DBL2b::ICAM-1domain interactions [24] is incomplete and may require further refinement to which our data will contribute. We cannot exclude though the possibility that variant DBLb domains may interact differently with the ICAM-1 receptor and future studies on structure-functional relationships for other ICAM1binding PfEMP1 domains (including experimental crystallographic data) will contribute to a better understanding of the precise molecular mechanisms of interactions. In our studies the Ala286Tyr (numbering according to [24]) amino acid residue substitution that has currently been found only in ICAM-1 nonbinding domains does not reduce binding of DBL2b PF11_0521 domain. Point mutations in a predicted conserved a-helix located in the N-terminal sub-domain were also neutral. Our experiments with anti-DBL2b PF11_0521 domain antibodies (summary shown in Table S1) demonstrate that only antibodies to the full-length domain, but not to truncated fragments missing the N-terminal sub-domain or to N-terminal sub-domain, can inhibit interaction with ICAM-1. Our data indicate that truncated variants are likely mis-or un-folded. In addition, the full-length DBL2b PF11_0521 domain binds the ICAM-1 receptor with high avidity in the low nM range. These data indicate that selection of the correct boundaries of PfEMP1 domains is extremely important in the design of immunogens that will elicit functional adhesioninhibitory antibodies.  Model [24] was visualized (A) and amino acid substitution in loop 4 (B) was modeled using Deep View Swiss-PDB viewer. In panel A loops 1 through 4 participating in interactions of DBLb domain with ICAM-1 are shown in red, the rest of the molecule in green, ICAM-1 molecule in yellow. Ala286 residue in loop 4 that is involved in contacts with ICAM-1 is shown in blue. Y motif is circled. In panel B amino acid residue 286 (Ala or Tyr) is shown in pink. Amino acid residues Asn (N) and Tyr (Y), which are located at the beginning and the end of the loop 4, are indicated. Residues in the vicinity of the position 286 are shown in spacefilling shape with CPK coloring. Substitution Ala286Tyr creates an additional hydrogen bond (arrowhead in panel C) between Tyr286 and Arg113 (numbers according to [24]). (PPTX) Figure S3 Predicted secondary structure of N-terminal sub-domain and amino acid residue mutations in the first a-helix of DBL2b PF11_0521 . ICAM-1 binding and nonbinding domains [20,21] are grouped. Color of amino acid residues: purple -conserved at least in ICAM-1 binding sequences; blue -semi-conserved; red -having significantly different physical-chemical character from the majority of amino acid residues in this position that may affect structure or/and function of the domain. Site-directed mutations: R23RA, A25RK. (PPTX)


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Scanning Electron Microscopic Studies of the Pecten Oculi in the Quail (Coturnix coturnix japonica)

The main purpose of this study is to extend the microscopic investigations of the pecten oculi in the quail in order to add some information on the unresolved functional anatomy of this unique avian organ. The pecten oculi of the quail was studied by scanning electron microscopy. Eighteen- to-twenty two highly vascularised accordion-like folds were joined apically by a heavily pigmented bridge of tissue, which holds the pecten in a fanlike shape, widest at the base. The structure of the double layered limiting membrane was recorded. The presence of hyalocytes with macrophage-like appearance was illustrated. It is assumed that the pecten oculi of the quail resembles that of the chicken. Illustrated morphological features of this species may add information on the active physiological role of the pecten. But still, the functional significance of this organ is a matter of controversies.

Introduction
The pecten oculi is a unique anatomical structure of the avian eye. Three types of pecten are recognized: the conical type (pecten oculi conicus) found only in kiwis, the vaned type (pecten oculi vanellus) found in the other extant Struthioniformes, and the tinamous and the pleated type (pecten oculi plicatus) found in all other birds [1]. The pecten has been frequently studied and extensive literature about pecten is available [2,3]. Consequently, many investigations were carried out by light and transmission electron microscopy, regarding the structure of the pecten in various avian species (Table 1). In the great majority of the studies, light and transmission electron microscopy have been used.
The morphology of the pecten in the quail (Coturnix coturnix japonica) has been studied by scanning (SEM) and transmission (TEM) electron microscopy [4]. Moreover, branching of vessels in pecten oculi has also been investigated by light microscopy and stereomicroscopy [5]. However some morphological features such as the hyalocytes or the structure of the pectin-vitreal limiting membrane have not been recorded.
The present work aims to provide some unexplored morphological data of the pecten of the quail with the use of scanning electron microscopy. In addition, a concise review and discussion aim to summarize the up to date knowledge, in order to throw light on the functional structure of this organ.

Material and Methods
In the present study, six adult healthy quails of both sexes were used. Prior to decapitation, the animals were deeply anesthetized by inhalation of diethyl ether. The ocular bulbs were immediately removed and opened to facilitate access of the fixative used for immersion fixation. Ten of them were fixed in a sodium cacodylate buffered solution of 2% glutaraldehyde and 2% paraformaldehyde. Subsequently, pieces of the pectens were washed four times, for 15 min each time, with sodium cacodylate buffer (pH 7.2), then transferred for 1 h to 1% OsO 4 , and dehydrated in graded acetone series. Tissues were critical point-dried in carbon dioxide (72-75 Barr), mounted onto stubs, and sputter coated with platinum and gold in a Bal-Tec sputter coater. Specimens were observed in a scanning electron microscope (JEOL, JSM 840).

Results
The pecten of the quail was a fan-like intraocular structure situated over the head of the optic nerve. It was trapezoidal shaped and black in color. Pleated type pecten oculi was comprising 18-22-folds which were joined apically on a fibrous bridge (Figures 1(a), 1(b), and 1(c)). The pleats were attached basally at intervals along the entire length of the linear portion of the optic disc (Figures 1(a) and 1(d)). The pectin was measured 4-5 mm in length at its base, whereas the bridge measured 2.5-3 mm. The mean height and width of pleats were 1 mm and 100 m, respectively. A superficial membrane surrounding the pecten oculi was separating it from the vitreous body (Figure 2(a)). This structure was composed of two different laminae (Figure 2(b)). The basal lamina surrounded each blood vessel. It was attached on the outer aspect of the blood vessels and contained a large number of delicate reticular microfibrils (Figure 2(b)). The outermost had the appearance of amorphous substance (Figure 2(b)). The surface of the pleats showed a dense vascular network with branching and anastomosing vessels. Within this network extravascular spheroidal bodies were observed and identified as pigmented cells (Figure 3(a)). On the pectineal surface and especially on the pleats, amoeboid phagocyte-like cells, the hyalocytes were found (Figure 3(a)). These cells were not observed at the bridge of the pecten. They were seen isolated, double, or forming clusters both in the external and internal faces of the pleats. The hyalocytes had various shape, size, and number of cytoplasmatic processes. Their length varied from 6 m to 10 m whereas their width was 4-5 m. Their external morphology exhibited undulating membrane with filopodia ( Figure 3(b)). The transverse section of the vascular network of the pecten revealed the profile of numerous capillaries with diameter ranging from 5 to 10 m. Among them, each fold contained two or more larger blood vessels, which were difficult to differentiate as either arterioles or venules (Figure 4). Their diameter frequently exceeded the 20 m.

Discussion
The pecten oculi of the quail (Coturnix coturnix japonica), in concert with other avian species, is a pigmented highly vascular, fanlike structure situated over the head of the optic nerve and projecting out into the vitreous. My observations Anatomy Research International are in agreement with previous studies in the quail [4,5]. The morphometrical data of this study are in the same range with that of Orhan et al. [5]. The general morphology of the quail's pecten resembles that of chicken [6][7][8].
The limiting membrane which covers the pecten has been illustrated for the first time in terms of SEM. Besides the numerous morphological investigations of the avian pecten, some of them have illustrated the limiting membrane by means of TEM [7,9]. Our observations are similar to reports of chicken [7] and pigeons [9]. The authors demonstrated the double layered limiting membrane by TEM. The double layer constitutes a barrier between the pecten and the vitreous body.
The presence of hyalocytes on the pectineal surface of the quail has been demonstrated for the first time by means of SEM, in the present study. Previous investigations in the developing quail revealed the phagocytic activity of the hyalocytes [10]. These specialized cells have also been observed  in the chicken [8], the mallard [11], and the budgerigar [12]. In contrary, they have not been observed in many other avian species [2,9,13]. Fischlschweiger and O'Rahilly [7] described with means of TEM the presence of cells on the external aspect of the covering membrane. These cells, termed "peripectinate cells, " were thought by the authors to be a special type of astroglia. As these cells display phagocytosis and from their histochemistry, it has been concluded that the hyalocytes are a subtype of macrophage [8]. My observations agree with the positive relationship between the distribution of hyalocytes and blood vessels in the chicken [8]. In fact, the hyalocytes were numerous on the free surface of the pleats excluding the bridge in which the relatively fewer vessels were located in the deep portion. Llombart et al. [10] proposed that hyalocytes might prevent the movement of macromolecules into the retina by their macrophagic activity. The presence of pigmented cells is a constant feature of all pectens recorded to date. In all species described, there is a marked increase in melanocytes in the apex or bridge region of the pecten [2]. Overlying and within the vascular network, a close association between blood vessels and pigmented cells is evident. This association may serve as a protective mechanism of the vessels against the effects of ultraviolet light [12,14].
Fine structural studies to date indicate that while the pleated pecten was widespread and essentially similar in most species, there were some variations in such parameters as shape, size, number of folds, and the thickness of capillary basal laminae [15]. The number of pleats varies among the species which possess a pleated type pecten (Table 1). SEM studies showed the approximate number of pleats was [18][19][20][21][22] in the chicken [6,23]. In the present study the number ranged from 18 to 22 similar to chicken and to quail [4,5]. Many attempts have been made to correlate the number of pleats with the function of the pecten. From a survey of the existing literature (Table 1) no correlation could be found regarding the number and the zoological classification of the birds. The number of pleats in the quail is similar to the fowl [6,23]. Both of them have a zoological closeness since they belong to the same family of Phasianidae. On the other hand, the budgerigar [12] and the Australian galah [21] which belong to the family of Psittacidae have 10-12 and 20-25 pleats, respectively. The size of the pecten and the number of pleats do not appear to relate directly to the size of the eye itself. On the other hand, the size of the pecten and the number of pleats seem to pertain to the behavior of the bird in relation to its general activity and its environmental lighting. Furthermore, these variations seem to correlate with the diurnal activity and/or visual requirements of the species (Table 1). The same deduction has been recorded by Pettigrew [31] who compared the number of pectineal pleats of 8 diurnal and 8 nocturnal birds. However this correlation may not be evidential; for example, the emu has a pecten with 3-4 pleats [20].
Despite the numerous publications, the functional significance of the pecten remains controversial. It is more than a century ago when the function of the pecten has challenged the imagination of investigators. The pecten according to numerous authors is mainly concerned with the nourishment of the avascular avian retina [17,20]. The intriguingly dense meshwork of capillaries points to erectile tissue of the pecten which potentially could serve as sun shade [27]. The double limiting membrane provides the mechanical support of the pecten and a barrier between the pecten capillaries and the hyaloid body. In the same capillaries, blood which is circulated, could serve as carrier of various sensory molecules. The hyalocytes probably serve as sweepers of unwanted material within the hyaloid body [8]. Frost and Mouritsen [32] claimed that the pecten could possibly function as an "ocular sextant. " Maybe the pecten could potentially be engaged in avian navigation.

Conclusions
The purpose of the present study was to give some additional information on the morphology of the avian pecten and to explore possible relations between the structure and function of this organ. The appearance of the hyalocytes, as well as the illustration of the bilaminar structure of the covering membrane, was demonstrated for the first time. These morphological characteristics add information on the structure of the pecten oculi. However, there is need for a further approach to throw light in this enigmatic organ.