Alzheimer’s Disease (AD) is a neurodegenerative disorder related with the increase of age and it is the main cause of dementia in the world. AD affects cognitive functions, such as memory, with an intensity that leads to several functional losses. The continuous increase of AD incidence demands for an urgent development of effective therapeutic strategies. Despite the extensive research on this disease, only a few drugs able to delay the progression of the disease are currently available. In the last years, several compounds with pharmacological activities isolated from plants, animals and microorganisms, revealed to have beneficial effects for the treatment of AD, targeting different pathological mechanisms. Thus, a wide range of natural compounds may play a relevant role in the prevention of AD and have proven to be efficient in different preclinical and clinical studies. This work aims to review the natural compounds that until this date were described as having significant benefits for this neurological disease, focusing on studies that present clinical trials. Neurodegenerative diseases induce alterations in the central nervous system with psychological and physiological negative effects . AlzheimAβ derives from the amyloid precursor protein (APP), proteolytic cleavage of amyloid precursor protein (APP), an integral membrane protein that possesses the general properties of a cell surface receptor , by the Therefore, it is fundamental to seek for new strategies for AD therapy ,20,21,22In this review, the natural compounds already in clinical trials phase are described and the reported results are presented and discussed. Other natural compounds with known potentially beneficial effects in AD in a preclinical development stage with in vitro and in vivo studies are also described. For preclinical studies, only the most recent reported works are cited. The systematic literature search was conducted using PubMed, Science direct, Google Scholar, Scopus and Web of Science as online databases until April 2019. Only papers written in English were considered with unlimited publication date.Natural compounds are an emerging approach for AD therapy. For the assessment of their therapeutic efficiency and potential side effects, human trials have been performed in the last years. The first natural product studied in a clinical trial was nicotine in 1992. However, no clinical trials were performed in the last two decades for this molecule. During the 90s, several other compounds were studied in clinical trials for AD therapy, such as vitamins. These molecules are still being tested in human trials up until this date. In the last years, other natural compounds are gaining interest by the scientific community and have achieved the clinical trials phase, such as bryostatin, which effects started to be evaluated in humans in 2017. A detailed report of these findings is described below. The natural compounds were divided into two groups: bioactive compounds and natural extracts, and they are summarized in Vitamins have been described as therapeutic compounds for AD. Among them, vitamin C, E and D have aroused great interest. Vitamin C A is founOn the other hand, vitamin E, which is present in several fruits and vegetables B, also s3, 2, Other vitamin with reported beneficial effects for AD, is vitamin D. Adding to several benefits of vitamin D , its theClinical trials revealed that vitamin D increased plasma Aβ in mild cognitive impairment patients, suggesting a reduction in Aβ levels in the brain. In fact, Miller et al. (2016) studied the effect of vitamin D supplementation on the plasma levels of Aβ in eight patients over 60 years old in a pilot study. Patients were randomly divided in two groups, treatment and placebo groups. Patients from the treatment group were administered with 50,000 IU per week for eight weeks. The obtained results showed that vitamin D intake increased plasma Aβ levels, suggesting a decrease in Aβ brain levels .n = 47) were orally supplemented with vitamin D at 50,000 IU once a week for six weeks, followed by 1500–2000 IU daily for 18 months. The obtained results showed that lymphocyte susceptibility to death, Aβ plasma levels and cognitive status improved after six months of vitamin D supplementation in cognitive impairment patients, but not in very early AD patients. Thus, supplementation with vitamin D proved to be beneficial in cognitive impairment patients. The lack of effects in very early AD patients suggest that vitamin D intake is not able to delay the progression of the disease in a more advanced stage [SanMartin et al. (2017) evaluated the role of vitamin D in the Aβ clearance from the brain. Patients with mild cognitive impairment and very early AD (ed stage .n = 8), or memantine alone (n = 18), or vitamin D alone (n = 17). Patients were administered with drugs for 24 weeks. Memantine was administered orally at 5 mg per week for the first four weeks and then 20 mg per day for the rest of the trial. Patients received a drinking solution of vitamin D at 100,000 IU every four weeks. After the study, patients co-treated with memantine and vitamin D showed better cognitive performance than patients treated with vitamin D or memantine alone [Co-therapy with vitamin D and other molecules for AD therapy has also been explored in clinical trials. In fact, Annweiler et al. (2012) conducted a double-blind, placebo-controlled pilot trial with 43 white patients over 60 years with moderate AD symptoms . The maine alone .n = 78) received placebo or daily supplement containing 800 IU of vitamin E, 500 mg of vitamin D, 900 mg of α-lipoic acid and 400 mg of coenzyme Q for 16 weeks. The attained results showed that the co-supplementation did not affect amyloid or tau levels, but a reduction on levels of an oxidative stress biomarker, the cerebrospinal fluid F2-isoprostane, was verified.Co-supplementation with vitamin D and other natural compounds was also studied in clinical trials. In fact, Galasko et al. (2012) conducted a double-blind, placebo-controlled clinical trial to evaluate what antioxidant supplementation affected the levels of AD’s histopathological marks, such as Aβ peptide and tau protein . PatientAlso, co-supplementation with multivitamins was evaluated in clinical trials. In fact, Kontush et al. (2001) evaluated the efficiency of supplementation with both vitamin E and vitamin C to decrease oxidation of lipoproteins in AD patients . Lipid oCo-therapy of different drugs with vitamin E was also studied in clinical trials. Sano et al. (1997) evaluated the effects of vitamin E and selegiline co-administration . Selegiln = 769) were randomly divided in three groups, placebo, vitamin E alone or donepezil alone. The daily dose of vitamin E was 1000 IU, and after six weeks the dose was increased to 2000 IU, for five years. Vitamin E proved to not be able to delay the disease progression. The combined effect of donepezil and vitamin E was also studied. Donepezil is a drug used for AD therapy to control the symptoms. To compare the effects of this drug with vitamin E on the outcome effects on patients with mild cognitive impairment, a double-blind, placebo-controlled clinical trial was conducted by Petersen et al. (2005) . PatientDysken et al. (2014) studied the combination effects of vitamin E and memantine . For thaKryscio et al. (2017) intended to assess if vitamin E and selenium intake could prevent dementia in healthy men over 60 . AlthougDocosahexaenoic acid (DHA) is a polyunsaturated fatty acid from marine fish and algae  and its The effects of supplementation with DHA in AD patients were studied in different clinical trials. In fact, Freund-Levi et al. (2006) conducted a double-blind, placebo-controlled clinical trial with 204 AD patients . The maiQuinn et al. (2010) conducted a double-blind, placebo-controlled clinical trial to evaluate the efficacy of supplementation with DHA on the cognitive and functional decline in AD patients . A dailyThe same group conducted a double-blind, placebo-controlled, clinical study in the same year to evaluate the ability of DHA to improve the cognitive functions of 485 participants with age-related cognitive decline . The subn = 36) were randomly divided in two groups, placebo and treatment group. The treatment group was orally administered with 430 g of DHA three times a day, for one year. No significant side effects were verified, suggesting the potential of DHA to improve memory functions. However, studies with more patients and longer intervention periods, are necessary to define the optimal dosage.Lee et al. (2013) studied the effects of DHA administration using fish oil on the cognitive function in patients over 60 diagnosed with mild cognitive impairment . The parGrateloupia livida and its structural formula is presented in Homotaurine, also known as tramiprosate, is an aminosulfonate metabolite extracted from marine red alga Aisen et al. (2011) conducted a phase III double-blind, placebo-controlled trial with 1052 patients with mild to moderate AD symptoms to evaluate the effect of homotaurine in slowing AD progression ,49. ThisThe safety and tolerability of this compound administered to 58 patients with mild to moderate AD symptoms, were studied previously in a phase II clinical trial conducted by the same group . PatientMartorana et al. (2014) conducted a study with 10 patients with mild cognitive impairment with ages between 59 and 74 . The parHuperzia serrata (Thunb.) Trevis. (Lycopodiaceae) and its structural formula is presented in Huperzine A is isolated from Xu et al. (1995) evaluated the efficacy and safety of huperzine A in AD patients. Four tablets of huperzine A (200 µg) or placebo were administered orally to 103 patients, twice a day, for eight weeks . The resTo further compare the efficacy and safety of huperzine A administered into capsules and tablets in AD patients, the same group conducted a new trial four years later . In thisLater, Rafii et al. (2011) studied the safety and efficacy of two concentrations of huperzine A, 200 and 400 µg twice a day, in patients with mild to moderate AD in a phase II clinical trial . Placebobryozoan Bugula neritina [Bryostatin is a macrolide lactone extracted from neritina . The strneritina . Also, bneritina .2 was administered to six patients, while three patients received placebo. Bryostatin proved to improve cognitive functions and to be safe and well tolerated. Recently, Nelson et al. (2017) evaluated the safety, tolerability and effects on cognitive function of bryostatin on AD patients in a phase II clinical trial . A singlAnother phase II clinical trial was performed with the same goals . Farlow Melatonin is collected from animals, plants, fungi and bacteria and its structural formula is presented in Brusco et al. (1998) evaluated the efficacy of melatonin in monozygotic twins with AD, with similar cognitive and neuropsychologic impairments . Only onLater, the same group studied the effect of melatonin in cognitive dysfunctions of 14 AD patients . The patThe same results were obtained by Furio et al. (2007) that performed a clinical trial with 50 outpatients diagnosed with mild cognitive impairment, where half of patients received 3 to 9 mg of melatonin for 9 to 18 months . Wade et al. (2014) investigated the ability of 2 mg of melatonin to improve the cognitive functions of patients with mild to moderate AD . MelatonVitis vinifera L. (Vitaceae)) and red wine and its structural formula is presented in Resveratrol is a naturally occurring non-flavonoid polyphenol present in grapes  performed a phase 2 clinical trial for 52 weeks in mild to moderate AD patients. The group studied the safety, tolerability and the ability of resveratrol to reduce the biomarkers of the disease (Aβ and tau). Here, 119 individuals were orally administered once a day with placebo or 500 mg of resveratrol, with an increase of 500 mg each 13 weeks. Although this study suggests that resveratrol can cross the blood-brain barrier (BBB), the results were not satisfactory. Besides inducing some side effects like nausea, diarrhea, and weight loss, the brain volume and biomarkers levels were lower in the placebo group than resveratrol group .Recently, Zhu et al. (2018) evaluated the safety, tolerability and efficacy of a mixture containing 5 mg of resveratrol, 5 g dextrose and 5 g of malate. Fifteen mL of the mixture or placebo were orally administered twice a day to 39 patients with mild to moderate AD for one year. The administration was done together with an 8 oz glass of commercial grape juice. The results revealed that the preparation was safe and well tolerated. However, no evidence was observed concerning the efficacy of the product for AD therapy .Nicotiana tabacum L., Solanaceae) and its structural formula is presented Nicotine is extracted from the tobacco plant leaves  studied the effect of nicotine on AD patients . Three aThe effect of nicotine on behaviour, cognition, and physiology of six AD patients was evaluated in a pilot study proposed by Wilson et al. (1995) . PlaceboThe clinical and neuropsychological effects of nicotine was evaluated in eight AD patients by White et al. (1999) . TransdeCurcuma longa L. (Zingiberaceae) and its structural formula is presented in Curcumin is an active component founded in the root of Baum et al. (2008) performed a clinical trial to study the safety of curcumin on AD patients . For sixGinkgo biloba L., Ginkgoaceae) has been studied as therapeutic drug for AD and other neurological diseases therapy. In vitro evidence revealed that ginkgo biloba extract can prevent Aβ aggregation, decrease Aβ fibrillogenesis and destabilize preformed fibril [Ginkgo biloba  were considered in this study. Patients received 240 mg of extract or placebo once daily for 24 weeks. The treatment with gingko biloba was safe and improved the neuropsychiatric symptoms, which include apathy, irritability, depression, among others. Several clinical trials have been carried out in the last 10 years to test the viability of the compound in treating patients with dementia. Bachinskaya et al. (2011) examined the effect of gingko biloba extract EGb 761dementia ,68. OutpAlso, with the same conditions, Herrschaft et al. (2012) revealed that the treatment with gingko biloba improved the cognition and the life quality of patients . n = 333) or vascular dementia (n = 71). In addition to confirming the improvement of neuropsychiatric symptoms observed in the previous trial, the extract improved the cognitive functions and functional abilities of patients. Ihl et al. (2012) performed a similar 24-week randomised controlled trial involving 404 outpatients . Patient® or placebo for 24 weeks. The trial proved that the extract improved the neuropsychiatric symptoms and cognitive functions of patients. Also, the extract was safe and well tolerated. Taking together, the last clinical trials proved that a 240 mg daily dose of ginkgo biloba extract is safe in the treatment of dementia.Gavrilova et al. (2014) also conducted a clinical trial to study the effects of gingko biloba in neuropsychiatric symptoms and cognition in 160 patients with mild cognitive impairment . The patCrocus sativus L., Iridaceae) is a stem-less herb with antioxidant [Saffron  evaluated the efficacy of 30 mg saffron in the treatment of mild to moderate AD . SaffronMelissa officinalis L., Lamiaceae) from the mint family that is native to Europe with antioxidant activity in vitro [Melissa officinalis extract on patients with mild to moderate AD, Akhondzadeh et al. (2013) administered to 40 patients 60 drops of extract or placebo, for four months [Melissa officinalis extract ameliorated the cognition and agitation of AD patients. Lemon balm (in vitro . In vivoin vitro . To asser months . The resCamellia sinensis (L.) Kuntze, Theaceae) from steaming and drying of leaves of the Camellia sinensis plant proved to be a rich source of antioxidants in in vivo studies [Green tea  developed a clinical trial with 30 patients to study the antioxidant activity of green tea in patients with severe AD and its ability to improve cognitive functions . PatientCarica papaya L., Caricaceae) is a fruit often used in medicine that has amino acids, β-carotene, oligosaccharides and vitamins, with benefits in AD. Papaya (n = 20) received 4.5 g of extract daily for six months, while the 12 controls did not receive any treatment. The results showed that the supplementation with fermented papaya powder reduced the ROS generation and nitric oxide production in AD patients, with no significant changes in controls. Thus, the papaya can be used as antioxidant in the AD therapy.A clinical trial performed by Barbagallo et al. (2015) studied the antioxidant activity of fermented papaya powder extract in AD patients . AD patiSalvia officinalis L., Lamiaceae) is a medicinal plant with a long-standing reputation in European medical herbalism due to its anti-inflammatory and antioxidant properties observed in vivo [Sage  developed a clinical trial to evaluate the efficacy and safety of erate AD . PatientCocos nucifera L., Arecaceae) demonstrated to be able to reduce the Aβ deposition and aggregation and the oxidative stress in a transgenic Caenorhabditis elegans AD model [Coconut  performed a clinical trial with 44 AD patients . Half ofMalus domestica Borkh., Rosaceae) showed to be a promising approach to prevent AD. In vivo evidence demonstrated that the apple extract prevents the oxidative stress and reduces the Aβ levels, improving the memory of AD rats [Apple  performed an open-label pilot clinical trial with 21 patients with moderate to severe AD . The autVaccinium myrtillus L., Ericaceae) is a fruit composed by several polyphenols named anthocyanins, with antioxidant [Blueberry  evaluated the effects of daily administration of wild blueberry juice in a group of nine elderly subjects with early memory failures . The daiColostrinin, a milk form produced by mammary glands , presentThe effect of colostrinin on AD patients was studied in a clinical trial conducted by Szaniszlo et al. (2009) . PatientBesides the natural compounds that have been studied in clinical trials, several other products have proved to have a potential beneficial effect in AD therapy in a preclinical stage, namely in in vivo studies. The preclinical phase involving in vivo studies is conducted to assess if the new compounds are safe and effective, before they can proceed to the clinical trials phase. A detailed report of animal studies results is described below. The natural compounds were divided into two groups: bioactive compounds and natural extracts and organized by the number of mechanisms associated with AD therapy, from the highest to the least.Epigallocatechin gallate (EGCG) is a polyphenol found in green tea with several neuroprotective effects in AD. In vivo evidence suggests that EGCG decreased β- and γ-secretase actions and enhanced the α-secretase activity, leading to the decrease of Aβ levels improving the memory . BesidesRetinoic acid is a terpenoid and a metabolite of vitamin A. In vitro studies revealed that retinoic acid inhibited Aβ fibrils formation and their extension and destabilized Aβ fibrils . In vitrCaffeine is perhaps the most consumed psychoactive compound. It is present in the coffee bean, but it can be also found in some teas, cocoa drinks, candy bars, among other herbs. In vivo studies suggest that caffeine reduced the β-secretase and γ-secretase levels, decreasing the Aβ production . An in vScutellaria baicalensis Georgi (Lamiaceae). In vitro studies suggested that baicalein inhibits the ROS production, reducing the oxidative stress [Baicalein is a naturally occurring flavonoid from the roots of e stress . In vitre stress . In vivoe stress ,183. AlsBerberine is an isoquinoline alkaloid found in rhizoma coptidis, an herb frequently used in Chinese herbal medicine. In vivo evidence suggests that berberine inhibited the β-secretase expression, reducing the Aβ production. Also, berberine stimulated the Aβ clearance and inhibited the Aβ plaque deposition and hyperphosphorylation of APP and tau . BerberiKaempferol is a polyphenolic flavonoid found in fruits, vegetables and herbs. In vivo studies proved its antioxidant effect, improving the learning and memory of a transgenic drosophila AD model . Also, iQuercetin is a flavonol, naturally occurring polyphenolic compounds present in fruits, vegetables and herbs. In vivo studies showed that quercetin improved the memory and cognitive impairments of an AD model and reduced the oxidative stress . MoreoveRhus succedanea L. (Anacardiaceae) and also found in some fruits and vegetables. Fisetin proved to inhibit Aβ aggregation in vivo [Fisetin is a flavonoid extracted from  in vivo  and fibr in vivo , reducin in vivo . Also, a in vivo . Additio in vivo  and redu in vivo .Oleuropein is a polyphenol present in extra virgin olive oil with antioxidant  and antiTannic acid is a polyphenol found in herbs and fruits. An in vivo experiment showed that tannic acid is a natural inhibitor of β-secretase with anti-inflammatory properties, preventing the cognitive impairment of AD mice . One in Crocin is a carotenoid mainly found in the stigma of saffron flower. In vitro experiments showed that crocin inhibits the Aβ fibril formation  through Epicatechin represents one of the antioxidants from the flavonoids family. High amounts of this compound can be found in cocoa beans, green tea and grapes. In vivo data showed that epicatechin has antioxidant  and antiGallic acid is a phenolic acid present in fruits, vegetables and herbs. Gallic acid proved to have antioxidant  and antiFerulic acid is a phenolic compound naturally present in numerous fruits and vegetables. In vivo results revealed that ferulic acid is an antioxidant  and antiRutin is a bioflavonoid extracted from some vegetables and fruits. This product is a glycoside of the flavonoid quercetin with antioxidant and anti-inflammatory properties in vivo . The samSalvia miltiorrhiza Bunge (Lamiaceae) root. In vivo experiments showed a strong antioxidant and anti-inflammatory activities, improving the memory and learning of an AD mouse model [Salvianolic acid B is a phenylpropanol founded in the se model . Also, sse model . Anotherse model .Myricetin is a flavonoid extracted from several fruits, vegetables and herbs. In vitro proofs showed that myricetin prevents Aβ aggregation and consequent fibrillation ,230 due Citrus junos Siebold ex Tanaka, Rutaceae. An in vitro study revealed that naringenin inhibited the APP and β-secretase activity and reduced the levels of phosphorylated tau [Naringenin is a natural compound present in citrus fruits and tomatoes. It is the major flavanone constituent found in ated tau . As resuated tau . In vivoated tau  and antiated tau .Luteolin, a polyphenol flavonoid found in fruits, vegetables and herbs, exhibits potent anti-inflammatory activity in vitro  and antiAsiatic acid is a pentacyclic triterpene found in plants. Asiatic acid demonstrates an ability to inhibit the β-secretase and increase the α-secretase activity in vitro. Also, it demonstrates an ability to activate Aβ clearance , which ePueraria lobata (Willd.) Ohwi (Leguminosae) used to treat some diseases. In vivo studies found that puerarin inhibited the tau phosphorylation and reduced Aβ levels, ameliorating the spatial learning and memory in an AD mice model [Puerarin is an isoflavanone glycoside isolated from ce model . The bence model  and neurce model . Oleocanthal is one of the main active components of extra virgin olive oil. In vitro evidence suggests that this compound changes the structure of tau protein, inhibiting its aggregation  and fibrVitis vinifera L., Vitaceae. In vitro evidence proved the anti-inflammatory [Viniferin (trans ε-viniferin) is a polyphenol present in a variety of vines, including ammatory  and antiammatory  activitiammatory  and inhiammatory .Cornus florida L. (Cornaceae) and coconut palm Cocos nucifera L. (Arecaceae). An in vivo study showed that this compound decreases the Aβ levels and inhibits the Aβ aggregation, improving the memory of AD rat model [Scyllo-inositol, also known as scyllo-cyclohexanehexol, is one of the stereoisomers of inositol, found in dogwood at model . In vitrat model .Magnolia officinalis Rehder & E.H.Wilson, Magnoliaceae. In vivo evidence suggested that honokiol is an antioxidant [Honokiol is a poly-phenolic product found in ioxidant  and antiioxidant . In vivoioxidant . As resuioxidant .Apigenin is a flavonoid found in plants, fruits and vegetables. Numerous in vitro and in vivo works showed its anti-inflammatory  and antiCaffeic acid is a phenolic acid present in food, beverages and Chinese herbal medicines with antioxidant and anti-inflammatory properties in vivo. This compound improved the learning of AD rat models . In vitrβ-carotene belongs to the carotenoid family. One in vitro study reported that β-carotene has an anti-aggregation activity and destabilizes Aβ . AnotherRosmarinic acid is a phenolic carboxylic acid found in rosemary, lemon balm and peppermint, among others. An in vivo study proved that this compound has antioxidant properties, protecting an AD mouse model against memory deficits . Also, rLarrea divaricata Cav. (Zygophyllaceae) with in vivo antioxidant properties [Nordihydroguaiaretic acid (NDGA) is a compound found in operties . An in voperties .Cnidium monnieri (L.) Cusson (Apiaceae). An in vivo study showed that this compound significantly enhanced the memory of an AD rat model, that can be linked to its antioxidant activity [Osthole is a coumarin isolated from activity  and withactivity . Also, iactivity .Punica granatum L. (Lythraceae). An in vitro study proved that this compound inhibits of β-secretase activity preventing neurotoxicity [Ellagic acid is a polyphenol extracted from toxicity . Ellagictoxicity .Glycine betaine is an organic osmolyte, which could be isolated from vegetables and marine products. In vivo evidence revealed that glycine betaine reduces tau hyperphosphorylation and Aβ production, improving memory deficits . Also, gHydroxytyrosol is a phenolic compound extracted from the olive leaf and oil. In vivo studies demonstrated that it is a compound with antioxidant and anti-inflammatory properties . Also, hl-theanine is an amino acid present in green tea. An in vivo work showed that l-theanine decreased the oxidative stress and the Aβ levels [β levels . Also, tβ levels .Alexandrium ostenfeldii and accumulate in shellfish. An in vitro study revealed that 13-desmethyl spirolide C is a spirolide that can reduce intracellular Aβ accumulation and hyperphosphorylated tau levels [13-Desmethyl spirolide C is a marine compound belonging to the cyclic imine group produced by the dinoflagellate u levels . The redu levels .Hibiscus vitifolius L.  and has been reported in in vivo experiments to exhibit anti-inflammatory [Gossypin is a flavonoid found in ammatory  and antiammatory .Gynostemma pentaphyllum (Thunb.) Makino (Cucurbitaceae) and they are reported in an in vivo study to be products with antioxidant and anti-inflammatory activities, improving the cognitive impairment [Gypenosides are triterpenoid saponins extracted from pairment .O-galloyl-β-d-glucopyranose (PGG) is a polyphenol and the main constituent of the Paeonia x suffruticosa Andrews (Paeoniaceae) root, a tree peony native to China and used in traditional medicine practices. In vivo experiments proved that PGG inhibits the Aβ oligomerization, which prevents Aβ fibril formation, resulting in the decrease of Aβ levels and improvement of memory. PGG is also able to promote the destabilization of Aβ fibrils [1,2,3,4,6-Penta- fibrils . Enoxaparin is a low molecular weight heparin present in the intestinal mucosa of pigs. Enoxaparin reduced the Aβ load through the decreasing of β-secretase activity . Also, eMaclura pomifera (Raf.) C. K. Schneid. (Moraceae), Maclura tinctoria (L.) D. Don ex Steud. (Moraceae) and leaves of Psidium guajava L. (Myrtaceae), promoted the inhibition of β-secretase activity in vitro [Morin, a natural flavonoid mainly found in in vitro . Besidesin vitro . Naringin is a flavonoid present in citrus fruits, namely in grapefruit. In vivo studies suggested that the antioxidant and anti-inflammatory activities of this compound improved the learning and memory of AD rats .Angelica sinensis (Oliv.) Diels (apiaceae) with antioxidant and anti-inflammatory activities in vivo. As a result, an improvement in learning and memory of AD rats was observed [Vanillic acid is a phenolic acid extracted from the plant observed .Punica granatum L. (Lythraceae)). In vivo studies suggest that punicalagin has potential as a nutritional preventive strategy in AD due to its anti-inflammatory activity. This natural product favors the anti-amylogenic route through the inhibition of β-secretase, reducing Aβ levels [Punicalagin is an ellagitannin found in the fruit peel of pomegranate  Scop. (Crassulaceae) that improved the learning and memory injuries in an AD rat model due to its antioxidant activity [Rhodosin is a flavonol extracted from the root of activity . Allium sativum L., Amaryllidaceae) is frequently used in culinary and medicine. Several studies showed that the administration of aged garlic extract significantly improves the memory deficit by several pathways. In vitro studies demonstrated that aged garlic extract has antioxidant properties [Garlic  is one of the most used spices and has been traditionally applied in the treatment of some diseases and their symptoms. Cinnamon extract is found to inhibit in vitro tau aggregation and promote the disassembly of tau filaments [Cinnamon  is the source of olive oil, one of the most important ingredients in the Mediterranean diet. In vivo studies showed that extra virgin olive oil ameliorated behavioural impairments. Also, the oil reduced the Aβ and phosphorylated tau levels [Olive  is a dried fruit composed by fatty acids, vitamins, alpha tocopherol, and polyphenols, in particular ellagic acid. An in vitro study showed that walnut extract inhibited the Aβ fibril formation through the inhibition of Aβ fibrillation, and also defibrillated Aβ fibrils [Walnut  are composed by several polyphenols including catechin, epicatechin, epigallocatechin and epicatechin gallate. In vivo studies have revealed that grape seed extract increases the memory performance and reduces ROS production, thereby protecting the central nervous system [Grapes  is a fruit with a variety of antioxidant polyphenols. Pomegranate juice reduced the Aβ levels and amyloid plaques in an AD mouse model, improving spatial learning and cognitive performance [Pomegranate  is a native American plant commonly used in traditional Chinese medicine. An in vivo study found that skullcap was able to protect hippocampal neurons against Aβ-induced damage through the attenuation of oxidative stress and neuroinflammation [Skullcap (ammation . Fragaria x ananassa (Weston) Duchesne, Rosaceae) is known to contain high phenolic contents. In vivo studies showed that strawberries have anti-inflammatory [Strawberry , an Asian and African plant, presents several nutrients, including β-carotene, vitamin C and E and phenols, including quercetin and kaempferol. In vivo studies showed that this plant improved the memory and learning due to its antioxidant activity [Moringa (activity .Besides the aforementioned natural compounds studied in human and animal studies, several other products have gained an increasing interest in scientific community for AD therapy. In fact, different compounds were tested in vitro and showed promising results. Some compounds proved to be efficient in preventing the formation of Aβ aggregates and disassembling Aβ fibrils, such as the case of tetracycline , methyl The reduction of Aβ levels can occur through changes in the structure of Aβ aggregates induced by natural compounds such as piceatannol . This prBesides these mechanisms, natural compounds can prevent AD progression by other mechanisms. For example, yessotoxin , gambierDespite the verified good outcomes, the study of some of these compounds was abandoned. For example, tetracycline was studied in 2001 but no more studies were reported for this compound. Also, for epicatechin gallate no studies were reported since 2003, and for retinal and retinol since 2004.Several bioactive compounds and natural extracts that were described herein to treat and prevent AD were revised and discussed. Until this date, most of the studied natural compounds are mainly derived from vegetable sources, with just a few molecules isolated from animals and marine organisms. Since AD is a multifactorial disorder, different therapeutic mechanisms were associated with these natural compounds. The approval process for a new compound to become clinically available is an extremely lengthy process, and it is divided into different phases. Before tests on humans, new compounds must be evaluated in preclinical studies. Several natural compounds proved to be promising for AD therapy in in vitro and in vivo studies, as discussed in this work. However, due to physiological differences between tested animals and humans, clinical trials are still necessary to validate the safety and efficacy of these compounds. Clinical studies are of outmost importance for the development of new therapeutic compounds, drugs and devices. Human studies allow to assess safety, tolerance and effective therapeutic doses for treating diseases. Some of the performed clinical trials described in this review did not show significant improvement in the delay or treatment of the symptoms. However, even if the trials do not exhibit positive outcomes, the obtained results can be still used to guide the scientists in the right path for drug discovery. Also, some of the conducted clinical trials with natural compounds for AD therapy, showed no conclusive results due to the limited size of samples. However, several compounds proved to be safe in human studies and were allowed to proceed to subsequent phases. To this date, homotaurine is the only compound that reached phase III of clinical trials for AD therapy.®, demonstrates beneficial effects in the patients. This product is already commercially available in some countries being partially financially supported by the public health care systems.Despite only a few natural products having been studied in clinical trials, numerous compounds proved to have beneficial properties in preclinical studies, as shown in Still, the neuroprotective effects of natural compounds depend of their ability to cross BBB. The low bioavailability of drugs and the difficulty to cross the BBB remains the major obstacles for the development of new therapies . Drug deAmong the studied natural compounds, only a small percentage have been encapsulated in DDS for brain targeting. Only the encapsulation of curcumin ,347,348,AD is a disabling disorder with a major negative impact on our current society. At this moment, no drugs have been developed to prevent or treat AD. The existing molecules only aim to control the symptoms. With the increase of average life expectancy, it is fundamental to discover and develop new molecules able to prevent and treat AD. Several natural products have proven to be promising for AD therapy in clinical and preclinical studies. Clinical trials have shown that several compounds appear to be effective for AD therapy, whereas others have failed in human trials. Natural compounds in earlier phases of research need further studies to uncover their therapeutic potential for AD.


While most studies addressed postsynaptic defects in the absence of MeCP2, we took advantage of an in vivo activity-paradigm (seizures), two models of MeCP2 deficiency, and neurobiological assays to reveal novel defects in presynaptic structural plasticity in the hippocampus in RTT rodent models. These approaches allowed us to determine that MeCP2 mutations alter presynaptic components, i.e., disrupts the plastic response of mossy fibers to synaptic activity and results in reduced axonal growth which is correlated with imbalanced trophic and guidance support, associated with aberrant expression of brain-derived neurotrophic factor and semaphorin 3F. Our results also revealed that adult-born granule cells recapitulate maturational defects that have been only shown at early postnatal ages. As these cells do not mature timely, they may not integrate properly into the adult hippocampal circuitry. Finally, we performed a hippocampal-dependent test that revealed defective spatial memory in these mice. Altogether, our studies establish a model that allows us to evaluate the effect of the manipulation of specific pathways involved in axonal guidance, synaptogenesis, or maturation in specific circuits and correlate it with changes in behavior. Understanding the mechanisms underlying the neuronal compromise caused by mutations in MeCP2 could provide information on the pathogenic mechanism of autistic spectrum disorders and improve our understanding of brain development and molecular basis of behavior.Methyl cytosine binding protein 2 (MeCP2) is a structural chromosomal protein involved in the regulation of gene expression. Mutations in the gene encoding MeCP2 result in Rett Syndrome (RTT), a pervasive neurodevelopmental disorder. RTT is one of few autism spectrum disorders whose cause was identified as a single gene mutation. Remarkably, abnormal levels of MeCP2 have been associated to other neurodevelopmental disorders, as well as neuropsychiatric disorders. Therefore, many studies have been oriented to investigate the role of MeCP2 in the nervous system. In the present work, we explore cellular and molecular mechanisms affecting synaptic plasticity events  Rett syndrome (OMIM #312750) is one of the few ASDs of monogenic origin that results in mental retardation, motor dysfunction, seizures, and features of autism. In 1999, the main cause of RTT was shown to be mutations in the MeCP2 . This prNumerous studies in animal models of RTT indicate that MeCP2 contributes to the formation and maintenance of neuronal connectivity; MeCP2 deficiency affects neuronal maturation , axonal in vitro MeCP2 regulates gene expression induced by neuronal activity; activity increases MeCP2 phosphorylation .in vivo, in real-time studies for understanding the molecular basis of behavior in several neurodevelopmental or neurocognitive disorders.We propose that this is a model to study the causal relationships between structural alterations in the hippocampus and cognitive alterations present in mouse models of RTT. These studies may lay the foundations to establish a system that allows evaluating the effect of manipulating specific pathways involved in neural connectivity in a specific circuit and correlating it with changes in behavior. Moreover, these results may serve useful for approaching We used two RTT mouse models for these studies. (1) MeCP2-308 model (MUT), these animals carry a premature stop codon at amino acid 308, generating a truncated MeCP2 protein that lacks the C-terminal region   and (2) in vivo stimulation paradigm. In the case of the KO, we used 6-weeks-old males, since the response to KA was already reported in the literature  injection of kainic acid . As a control, the same volume of sterile PBS was injected . Consideterature . It is iterature  while materature .After KA injection, seizure response was recorded, which became evident after 20 min of injection. From the moment of KA injection, the animals were individually housed in separate boxes and monitored for several hours. The severity of the seizure response was graded according to the following scale: 0, similar to control; 1, increased respiratory rate; 2, in a state of “freezing” and with erratic contractions of the body; 3, straight and stiff tail with or without shaking; 4, the front legs begin to tremble; 5, straight and stiff tail along with tremor of front legs (once); 6, continuously shows straight and stiff tail along with front leg tremor (more than twice); 7, complete clonic tonic seizures, jumps; 8, death .Two weeks after the KA injection, all animals were euthanized and perfused for morphometric analysis, according to F(1.12) = 1.228; p = 0.2895] and BDNF  in response to KA stimulation. These results demonstrate that in MUT MeCP2 animals an increase in neuronal activity was induced in response to KA administration, similar in magnitude to that induced in WT animals.To determine whether the lack of IPT growth in response to seizure activity in MeCP2 MUT mice was caused by defective neural activation in response to KA, the expression of activity-induced neuronal genes (Arc and BDNF) was evaluated. For this, new groups of mice from both RTT models were injected with KA and 6 h later, whole hippocampus were dissected from control and KA-injected MUT mice and WT littermates. The expression levels of Arc and BDNF were quantified by real-time RT-PCR. As shown in p < 0.05); and the levels of KA-induced Arc and BDNF expression in these animals were similar to the WTs  . KO mice the WTs . Neverth the WTs .F = 0.2665; p = 0.6197]. A significant increase in the number of dividing cells was observed in both WT and MUT KA-treated mice in comparison with their respective controls (p = 0.8169). To confirm that DG dividing cells were neurons, we performed double labeling IHC for BrdU combined with the neuronal marker tubulin β-III (Tuj1) or with an astroglial cell marker (GFAP). Most BrdU+ cells co-localized with the neuronal marker Tuj1, but not with GFAP. +) that remained 5 weeks after the induction of seizure activity. + cells per DG 8 days and 35 days after KA-induced seizures, when the new DG cells become mature neurons. At 35 days, BrdU+ cells from WT mice decrease about 60% and in MUT 71% respect to day 8; therefore, no significant differences were detected between the genotypes in terms of BrdU+ cell loss . Also, no differences were detected in the absolute number of BrdU+/DG cells between both genotypes at either 8 (p = 0.9987) or 35 (p = 0.8383) days after KA.It has been shown that the axons of adult-born neurons in DG contribute mainly to the plasticity of the IPT tract . Likewiscontrols . In addiThese observations suggest that the survival of the new DG neurons is not affected by MeCP2 mutation; therefore, we conclude that the lack of increase in the size of the IPT, registered 2 weeks after KA injection, was not due to a loss of new DG cells in MUT mice.+ NeuN+), the percentage of immature cells (BrdU+ DCX+) and at intermediate stage of maturation (BrdU+ DCX+ NeuN+). F = 13.73; p < 0.001]; the percentage of new DG cells that reached maturity was significantly lower in MUT mice . In addition, MUT mice showed a higher proportion of both immature cells  and cells in transit to maturation . Thus, MUT adult-born granule cells show abnormal maturation; immature cells accumulate at expense of lower numbers of mature cells.Previous work in the olfactory system and in the hippocampus from MeCP2-KO mice has reported a delay in neuronal maturation during early postnatal development, which is apparently compensated later on . Given tThese results indicate that mice carrying a mutated MeCP2 protein (MUT) show defective neuronal maturation in the adult hippocampus, similar to what was observed during early development in a different mouse model .Using the olfactory system and MeCP2-deficient mice, we have demonstrated that axonal guidance defects were accompanied by alterations in the expression of Sema 3F and its receptor complex (PlexinA3 and Neuropilin-2). It has been demonstrated that this axonal guidance molecule regulates axonal growth, guidance, and targeting of hippocampal MF, acting as an inhibitor of axonal growth when combined with its receptor complex present in axons and inducing growth cone collapse . ConsideMethyl-CpG 2 binding protein 2 MUT mice and WT littermates were injected with KA or PBS and 2 weeks later the hippocampus of the different experimental groups were dissected. mRNA expression levels of Sema 3F gene and their two receptors (PlxnA3 and Npn-2) were quantified by real-time RT-PCR. F = 4.7; p < 0.05]. A significant decrease of Sema3F expression was observed in WT mice + KA compared to WT controls, while in MUT mice no change was registered between controls and KA-treated mice. Likewise, the WT + KA mice presented significantly lower levels of Sema 3F compared to the MUT KA. For PlxnA3  = 1.327; p = 0.2687]. Although there were no changes in the expression of Npn-2 in response to KA treatment  = 8.072; p < 0.05].Regarding Sema 3F , consider PlxnA3 , no signreatment , we founSummarizing these observations, the lack of increase in IPT size after KA treatment, in the context of MeCP2 mutations, was accompanied by a deregulation in the expression levels of Sema 3F which, in concert with the receptor complex, exerts an important chemo-repulsive effect on hippocampal axons .in vitro, extends innervations of MF into CA3, and regulates its synaptic plasticity  = 10.61; p < 0.01]. WT animals showed a moderate but significant increase in BDNF expression, 2 weeks after injection with KA. In contrast, in MUT mice BDNF expression remained unchanged between the control and the KA-treated group. Also, BDNF levels in MUT+KA animals were markedly lower than those of WT + KA mice  = 11.58; p < 0.01]. While WT + KA showed a significant increase of pTrkB-positive cells in comparison with the controls, no significant differences were observed between treatments in MUT mice  in the dorsal region of MeCP2 MUT hippocampus and considering that this circuitry is mainly involved in the processing of spatial memory , we deciF = 0.744; p = 0.6150] ; “non-learners” were those animals that did not enter the escape box in any of the three training sessions. However, when we evaluated the escape latency of the animals that did learn, no significant differences were found in the acquisition between the groups [ 0.6150] . We founF = 14.81; p < 0.001]  = 22.7; p < 0.0001]. WT mice explored the target quadrant and the escape hole significantly more than MUT mice .in vivo, as a way to provide controlled stimulation conditions, leading to more homogeneous activity. We based our experimental design on several reports using KA injections in different Rett mouse models, considering that those studies have established most of the activity-dependent responses described in the field  model which better resembles the human pathology; these mice express a mutated protein, their clinical evolution is progressive, and they survive for over a year. This is important because it permits experiments at more advanced ages and also avoids the results may be masked by near-death physiological conditions, as occurs with most of the studies performed in MeCP2 KO models . The original characterization of this mouse model revealed that MeCP2 MUT animals develop spontaneous seizures from 5 months of age . HoweverHowever, we observed no change in the IPT volume in 9-week-old MUT mice , even th+) that remain 5 weeks in the DG after the induction of seizures activity was similar in WT and MUT mice  . HoweverOn the other hand, a body of evidence suggests that semaphorins are critical determinants of axonal growth and targeting during the development of the nervous system . Mice deIt has been suggested that the process of structural plasticity shown by MF in response to synaptic activity involves a tightly regulated balance between growth factors and axonal guidance molecules . Here weIn this study, we did not analyze the dendritic components of granule DG cells. However, in contrast to postmortem studies in RTT patients , the braper se is altered in patients, cognitive abnormalities are present in individuals with MeCP2 mutations, supporting the concept that the results obtained with the MeCP2-308 animal model would represent a correlate of cognitive deficits in RTT , which follows guidelines from the National Institute of Health.MB, MZ, and MF contributed to the acquisition, analysis, or interpretation of data for the work. SA, GVR, and GAR contributed to key techniques and critically revised the work for important intellectual content. AD contributed to the conception and design of the study, and wrote the first draft of the manuscript. All authors contributed to the manuscript revision, read, and approved the submitted version.GVR is currently employed by company Janssen Research & Development, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Diverticulosis of the colon is the most common condition in Western societies and it is the most common anatomic alteration of the human colon. Recurrent abdominal pain is experienced by about 20% of patients with diverticulosis, but the pathophysiologic mechanisms of its occurrence are not completely understood. In the last years, several fine papers have showed clearly the role of low-grade inflammation both in the occurrence of symptoms in people having diverticulosis, both in symptom persistence following acute diverticulitis, even if the evidence available is not so strong. We do not know yet what the trigger of this low-grade inflammation occurrence is. However, some preliminary evidence found colonic dysbiosis linked to low-grade inflammation and therefore to symptom occurrence in those patients. The aim of this paper is to summarize current evidences about the role of inflammation in symptom occurrence in symptomatic uncomplicated diverticular disease and in symptom persistence after an episode of acute diverticulitis. Diverticulosis of the colon is the most frequent anatomic alteration that can be detected during colonic exploration, and its incidence is increasing worldwide. It is a common finding especially in developed countries: by the age of 85, the large majority of the population living in developed countries will have developed colonic diverticula . Recent Most people with colonic diverticulosis remain asymptomatic. Just about one-fourth to one-fifth of those people will develop symptoms without complications, experiencing the so-called “Symptomatic Uncomplicated Diverticular Disease” (SUDD), and about 20-25% of them will ultimately develop diverticulitis (uncomplicated or complicated) –6.Although advancing age is obviously associated with diverticulosis, the occurrence of symptoms is probably not only linked to age but also to the prolonged time course, during which the colonic wall is exposed and makes the colon more susceptible to other pathogenetic factors.The pathogenesis of the disease is poorly understood and several etiological factors may play a role in its onset. In this paper, we focused on the roles of inflammation as part of the pathophysiology of diverticular disease.vasa recta [Diverticulosis is characterized by the presence of sac-like protrusions of mucosa and submucosa, called diverticula, which occur in the weakest point of the muscle layer. This occurs due to age-related degeneration of the mucosal wall and segmental increases in colonic pressure resulting in bulging at points of weakness, typically at the insertion of the sa recta .Although the underlying pathological mechanisms that cause the formation of colonic diverticula remain unclear, itis likely to be the result of complex interactions between genetic factors, age, diet, changes in colonic structure and colonic motility, and therefore colonic microbial imbalance .Inflammation probably does not play any role in the occurrence of colonic diverticulosis. Two European papers, published at the beginning of this decade, found that no signs of histological inflammation were found in people having colonic diverticulosis , 8. ThesAs stated, just one-fourth to one-fifth of patients with diverticulosis develop symptoms, the so-called Symptomatic Uncomplicated Diverticular Disease (SUDD). The pathogenetic mechanisms that lead to the occurrence of symptoms are still under debate, probably because there is still no consensus about the definition forms of the disease.p = 0.04) [SUDD is characterized by nonspecific attacks of abdominal pain without macroscopic evidence of an inflammatory process, with abdominal pain, bloating, and changes in bowel habits that may resemble IBS. As a consequence, several authors still talk about “IBS with diverticulosis.” For example, Jung et al. found that IBS occurs 4.7-fold more likely in patients after an episode of acute diverticulitis than controls , and mor = 0.04) .It is likely that those patients are suffering from SUDD than from “IBS with diverticulosis,” even when we describe symptoms' occurrence/persistence following an episode of acute diverticulitis. This is because IBS and SUDD are not the same clinical entity, as showed by clinical data currently available. IBS and SUDD do not share the same epidemiology  but onlyAs it is well known, acute diverticulitis (AD) shows significant sign of inflammation . However2-related enzyme in patients with AD. In particular, they observed a downregulation of cyclooxygenase-2/PGE2 and upregulation of 15-PGDH in the human colonic mucosa that may be predisposing factors for the development of acute DD [2 may predispose to AD occurrence. Moreover, we found that AD and Crohn's disease (CD) have a similar pattern of expression of basic fibroblast growth factor (bFGF), syndecan 1 (SD1), and tumor necrosis factor α (TNF α), supporting the hypothesis that inflammation may also activate the fibrosis process [Also, inflammation may play a role in the pathogenesis of the disease. Dai et al. showed a different expression of prostaglandin Eacute DD . Based o process .α, its overexpression in AD as well as CD is not regulated by the same gene superfamily 15 (TNFSF15), which is an immunoregulatory, antiangiogenic gene [p = 0.03) in all DD versus all control comparison. A second haplotype, rs6478108 (A), rs6478109 (G), rs7869487 (A), and rs4263839 (G), was also associated with DD in that cohort (p = 0.025). A third haplotype, rs6478108 (A), rs6478109 (G), rs7848647 (G), rs7869487 (A), and rs4263839 (G), was demonstrated in the DD < 55 years versus control > 55 years' comparison (p = 0.045). This study demonstrates clearly that distinct but overlapping TNFSF15 haplotypes can be found in diverticulitis patients versus healthy controls when compared with the known CD haplotype suggesting similar but distinct genetic predispositions [About the role of TNFnic gene , 20. A rositions . This stositions , triggerα, interleukin- (IL-) 6, and IL-1β levels were significantly higher and histology was significantly worse in patients having symptom persistence following severe AUD  [We know that up to 20% of patients complain of persistent abdominal pain after surgical treatment of diverticulitis , and thearisons) .p = 0.0004) [Persistent low-grade inflammation following an episode of AD may also explain the recurrence of AD. At the end of a 24-month follow-up period, we found that endoscopic inflammation was still detected in 27.67% of patients and active histological inflammation in 36.6% of patients following an episode of AD. Only the detection of endoscopic and of histological inflammation during the follow-up was a predictor of diverticulitis recurrence ( 0.0004) .Inflammation persistence may be therefore a risk factor for both symptoms' persistence and disease recurrence. Despite this, a recent review found mesalazine, an anti-inflammatory drug widely used in the treatment of Inflammatory Bowel Diseases, not superior to placebo in preventing diverticulitis recurrence . It is pAs stated, diverticulosis is merely the presence of colonic diverticula without symptoms or macroscopic/microscopic signs of inflammation. In this way, no signs of inflammation should be detected, both at endoscopic and also at histological assessment.A different scenario may be observed when we analyze patients with SUDD. In those patients, low-grade inflammation seems to really play a significant role in determining symptom occurrence and complication. The current evidences are as follows.p < 0.02).SUDD shows a significant microscopic inflammatory infiltrate. More than ten years ago, Narayan and Floch found a significant inflammatory infiltrate in SUDD patients compared with healthy controls . We asseα, a 17 kDa polypeptide produced by macrophages, lymphocytes, and natural killer cells, has been shown to play a major role in the inflammatory process, with high levels being found both in Inflammatory Bowel Disease (IBD) and in rheumatoid arthritis patients [α was assessed in those patients yet.TNF-patients –33. Hypoα expression was significantly higher not only in AUD than in HC (p = 0.0007) but also in SUDD than in HC (p = 0.0007) and in asymptomatic diverticulosis (p = 0.0001). Moreover, TNFα expression in AUD did not differ significantly from that of UC (p = 0.0678) and SCAD (p = 0.0610). Finally, it was significantly higher in UC, SCAD, and AUD than in SUDD  [α expression was significantly higher in SUDD than in asymptomatic diverticulosis (p = 0.04) [Assessing its mucosal expression on biopsy samples by reverse transcription polymerase chain reaction, we found that TNFctively) . The samp = 0.067 and SCADp < 0.05). Moreover, they found a slight increase of IL-10 release in SUDD and a significant IL-10 release in SUDD-AD than HC (p < 0.05) [p = 0.326) [IL-10 is an important anti-inflammatory mediator that suppresses the production of proinflammatory cytokines . Surpris < 0.05) . We rece= 0.326) .2 in colonic mucosa between acute diverticulitis and IBD [2 is the dominant prostaglandin in the colon and it is associated with colonic inflammation, but we also know that it has a protective mechanism in the mucosa of the gastrointestinal tract. Lower prostaglandin E2 level may decrease the normal protection of the mucosa, which is made more susceptible to luminal insults, thus creating a permissive environment for the development of acute diverticulitis.Thus, we can speculate that IL-10 expression in SUDD may be an attempt of the immune system to control a low-grade inflammation. This hypothesis seems to be confirmed by Dai et al., who found an inverse expression of prostaglandin E and IBD . We knowFecal calprotectin (FC) is a cytoplasmic antimicrobial compound prominent in granulocytes, monocytes, and macrophages, and it accounts for approximately 60% of the total cytosolic protein. It is released from cells during cell activation or death, and it is stable in feces for several days after excretion . This hap = n.s.). Higher FC values were found in AUD (p < 0.0005) and SUDD (p < 0.005) than in HC and IBS patients. Moreover, FC values correlated significantly with inflammatory infiltrate (p < 0.0005) and decreased to normal values after treatment both in AUD (p < 0.0005) and in SUDD (p < 0.005) [After a preliminary report describing an increase of FC expression in patients with “symptomatic diverticular disease” (no more information were provided by authors) , we perf< 0.005) . Althoug< 0.005)  and doesSeveral clinical trials in SUDD patients found 5-aminosalicilic acid  –45 effecSome studies tried to show that these are flaw evidences, since patients having diverticulosis and chronic abdominal symptoms do not show any evidence of inflammation . HoweverLow-grade inflammation may also interact with other pathogenetic mechanisms to lead symptom occurrence.p = 0.01); a higher but not significant expression of galanin 1 receptor (GALR1) in SUDD than asymptomatic patients was found [α, and an upregulation of the neuropeptide receptor NK1, suggests that SUDD patients may exhibit visceral hypersensitivity due to peripheral sensitization with both inflammatory and neurochemical factors playing a role.For example, visceral hypersensitivity has been suggested as a possible mechanism involved in causing symptoms in those patients , 49. Humas found  as well.β, Cossais et al. found very recently that S100β expression was increased in the submucosal and myenteric plexus of patients with DD compared with that in controls, whereas the expression of other glial factors remained unchanged. Moreover, this S100β overexpression was correlated to CD3+ lymphocytic infiltrates in patients with DD, whereas no correlation was observed in controls [Recent studies demonstrated that DD is associated with peculiar alterations of the enteric nervous system (ENS), encompassing myenteric oligoneuronal hypoganglionosis, decreased intramuscular nerve fibers, and altered neurochemical coding –53. Analcontrols .Proteobacteria than controls [Microbacteriaceae were the most significant species associated with the disease [Akkermansia muciniphila, a mucolytic bacteria living in the mucus barrier that degrades mucin for utilization as a carbon and nitrogen energy source [Roseburia hominis [A. muciniphila and R. hominis have a significant abundance in SUDD [It has been hypothesized that microbial imbalance, which is known as dysbiosis, may be the trigger of inflammation (and therefore symptoms) in people having diverticulosis . Danielscontrols , and Sch disease . Microbiy source  and prody source , is signy source . The sam hominis . In SUDD in SUDD –65, and  in SUDD –65.Diverticulosis is a common condition that sometimes becomes symptomatic and may lead to severe complications.The pathophysiologic hypotheses behind symptom occurrence in those patients have changed in the last years. Although most of the mechanisms leading to its occurrence remain to be elucidated, the role of low-grade inflammation seems to be a proven fact (Why inflammation occurs in only a minority of people having diverticulosis is still under debate, and it has to be further elucidated.


Lactation is at one point perilously near becoming a cancerous process if it is at all arrested”, Beatson, 1896. Most breast cancers arise from the milk-producing cells that are characterized by aberrant cellular, molecular, and epigenetic translation. By understanding the underlying molecular disruptions leading to the origin of cancer, we might be able to design novel strategies for more efficacious treatments or, ambitiously, divert the cancerous process. It is an established reality that full-term pregnancy in a young woman provides a lifetime reduction in breast cancer risk, whereas delay in full-term pregnancy increases short-term breast cancer risk and the probability of latent breast cancer development. Hormonal activation of the p53 protein (encode by the TP53 gene) in the mammary gland at a critical time in pregnancy has been identified as one of the most important determinants of whether the mammary gland develops latent breast cancer. This review discusses what is known about the protective influence of female hormones in young parous women, with a specific focus on the opportune role of wild-type p53 reprogramming in mammary cell differentiation. The importance of p53 as a protector or perpetrator in hormone-dependent breast cancer, resistance to treatment, and recurrence is also explored.“ Breast cancer is endemic worldwide, with a cumulative lifetime risk of 1:8 by age 70 ,3,4. AboTP53 gene) and female hormone regulation during the differentiation of the mammary gland in early and late pregnancies, which influences a woman’s susceptibility to breast cancer later in life [There is a very strong association between the function of the p53 tumor suppressor  and post-genetic alterations  during pregnancy are strongly implicated in breast cancer development and protection .Late-onset breast cancers are more likely to originate from ERα-positive luminal epithelial cells, or milk-producing cells, which have been exposed to years of fluctuating hormonal changes from puberty to pregnancy and menopause . As mentN-methylnitrosurea (MNU) or oral 7,12-dimethylbenz(a) anthracene (DMBA), with a near 100% latency period between 8 and 12 weeks [There is irrefutable, reproducible evidence from human studies and in rodent models that full-term and multiple pregnancies have a lifetime protective effect against breast cancer ,31,32. R12 weeks . Pioneer12 weeks ,39). Dur12 weeks . In preg12 weeks . An inte12 weeks , found t12 weeks ,42,43. A12 weeks . This ob12 weeks . ChromatFrom early studies in rodents, it was unclear whether completion of full differentiation per se (in the absence of estrogen and progesterone) reduced breast cancer risk. A few studies tested whether induction of full differentiation of the mammary gland was indeed the basis of protection against carcinogenicity ,14,45. PEarly studies, discussed in Breast tumors in rodents possess high similarity to ERα-positive and progesterone receptor (PR)-positive human breast tumors. Thus, rodent models have been key in our understanding of breast cancer development ,43. As eStudies, dating back to 1962, demonstrated that the female hormones estradiol-17β and progesterone administered to rats and mice in doses that mimicked pregnancy reduced the incidence of carcinogenicity and conferred long-term cancer protective effects; however, in these studies neither hormone individually was protective ,16,17,18In a study treating rats with estradiol-17β and progesterone for 21 days to mimic the parous protective effects against MNU, a set of 100 markers were identified, displaying persistent changes (upregulated) in the mammary gland compared to virgin rats . One of In general, the consensus from these studies, and many more not cited, is that short-term administration of estrogen and progesterone, that mimic pregnancy, can reduce malignant tumor formation and breast cancer latency. These changes are accompanied by persistent changes in the cellular and molecular profiles of the mammary cells, making the mammary gland less vulnerable to carcinogens or carcinogenic insults.Early studies by Russo and others suggested that human placental hormone chorionic gonadotropin (hCG) provided an inhibitory action, leading to reduction in breast cancer risk, as reviewed in reference . This loOverall, these studies are illustrative of many other works that explore the influence of the female hormone flux in pregnancy on breast cancer risk. They demonstrate that short-term administration of female hormones, whether it be estrogen and progesterone or hCG to induce mammary gland differentiation, has promise as a therapeutic protective intervention for breast cancer latency. Nevertheless, the challenge is when and how to administer preventative hormones. One of the major questions which still remains unanswered is: what are the molecular changes that govern persistent alterations in hormone-dependent signaling pathways that occur in young parous females and reduce lifetime breast cancer risk? The answer may be in p53’s function, as p53 is believed to be implicated in a number of steps relating to the risk and development of breast cancer.TP53 gene) has stereotypically been widely researched as the ultimate tumor suppressor protein important in driving cancer cell death through apoptosis or permanent irreversible cell senescence [For decades, the p53 protein , the homolog of the human p14ARF protein, led to immortalization of mammary epithelial cells . p19ARF A major breakthrough in supporting the role of p53 in governing parity-related changes in the mammary gland came from p53-null mammary gland transplantation studies . By tranA quote from Beatson’s seminal paper on carcinoma of the mamma (1896) states: “a reasonable ground for thinking the active processes seen in a cancerous tumor are best explained by regarding the epithelium of the part as having taken on the powers of the germinal epithelium” . A likelThe mammary cancer stem cell theory is based on a model whereby transformation of early progenitor cells or stem cells eventuate in latent tumorigenesis. A central event in the development of cancer is the deregulation of normal stem cell self-renewal, eventually leading to uncontrolled proliferation, aberrant differentiation, and generation of a heterogeneous breast cancer cell population ,136. AltWild-type p53 protein regulates p53 transcriptional programs in the repair of genetic alterations and also by preventing epigenetic abnormalities ,137. TovA major difference in the molecular characteristics of normal breast epithelial cells and breast cancer cells is that the majority (approximately 75–85%) of normal breast cells do not express ERα and PR, whereas the majority of breast cancer cells contain high levels of both hormone receptors (ERα+PR+ cells) . Normal In this section, we review the mounting evidence that p53 and ER activities are mutually regulated in breast cancer. Conceptually, a question worth asking is: is the loss of, or change in, p53 function associated with the signature of “paracrine-to-autocrine” response in the transition from normal to breast cancer cells? In This review would be amiss if we did not comment on the seminal findings of the epic Million Women Study by Beral and colleagues, investigating the use of hormone replacement therapy (HRT) in postmenopausal women . In thisTP53 gene that disrupt cell cycle inhibition are associated with the onset of more than 80% of all cancers [TP53 gene are not the main culprits in breast cancer development and progression. The majority of breast cancers occurring later in life  are hormone-responsive (ERα-positive) and, interestingly, harbor functional wild-type TP53 [The p53 tumor suppressor is intimately associated with inhibition of cancer and, in general, mutations in the  cancers ,148,149. cancers . This obype TP53 . In manyype TP53 ,153. Resype TP53 ,156,157.As mentioned in Although reactivation of the p53 signaling pathway has been considered as a good breast cancer treatment strategy, there is growing evidence at the molecular level to suggest an association between ERα and p53, which protects breast cancer cells from dying ,162,163.Our laboratory has demonstrated that activation of the p53 pathway in ERα-positive breast cancer cells rapidly, within hours, shuts down DNA replication, downregulates proteins involved in apoptosis, and increases the levels of proteins that are involved in normal breast cell metabolism ,97,98. OERβ [α and ERβ knockout (KO) mouse models [α and ERβ KO mouse models, ERα was shown to be essential for normal reproductive development [β-KO mice had decreased side branching and partial alveolar development, the mammary glands secreted milk normally, and mothers were able to nurse their young [In 1997, a second ER was identified, named ERβ ,169. TheERβ . The majERβ . Each ofe models . In seleelopment . In contir young . Unlike ir young . Conventir young . Alternair young . In two ir young ,178. Thuir young . Thus, tIn essence, the role of p53 as a protector or perpetrator in breast cancer may very well be dependent on the ratio of ERα and ERβ in breast cancer. In turn, the crosstalk between ERα, ERβ, and p53 has the potential to affect hormone-dependent breast cancers responsiveness to endocrine and other breast cancer therapies ,178. Beatson’s theory from his doctorate dissertation was that lactation was very close to being a cancerous process and, if arrested in its progression, could become carcinogenic, as cited in reference . In otheMCF-7 cells are derived from the luminal breast epithelial, which consists of cells that differentiate to become milk-producing cells. This cell line has provided vital information on hormone regulation of breast cancer and treatments to combat this disease. We have recently demonstrated that activation of p53 in MCF-7 breast epithelial cells initiated changes in cell morphology and cellular metabolism consistent with a differentiated phenotype and was most likely to be important in cell survival and recurrence in ERα-positive breast cancer cells ,97,98. TOur studies and the work of other groups have strongly suggested that re-activation of the p53 signaling pathway is strongly implicated in chemotherapy- and endocrine therapy-resistant breast cancers, especially in the most prevalent breast cancer sub-type (ERα+/p53+) ,98. ActiThe theory that the p53 protein suppresses cell cycle progression, induces resistance to apoptosis, and maintains the breast cancer cells viable through metabolic reprogramming ,98 is anIn summary, we do not know the extent of the function of p53 in the origin of latent hormone-responsive breast cancers, and this review does not delve into the many complexities of the normal p53 function. What is emerging from these cited reports and others is that p53 expression at a critical timing in young age pregnancy is pivotal in preventing mammary gland tumorigenesis. The underlying associated molecular changes could be permanent reprogramming of cellular events and/or DNA methylation, imprinting a protective signature and maintaining normal cell homeostasis. With older age pregnancies or in nulliparous women, accumulation of DNA mutations or other aberrations in the mammary gland abrogate this protective signature, and the cells are more susceptible to carcinogenic agents. Hormonal activation of the p53 in the mammary gland is implicated in the development of latent breast cancer. Confidence in the protective effect of p53 comes from p53null rodents, where, in the absence of p53, spontaneous tumors are found in the mammary glands, independent of age, as summarized in How can we utilize this knowledge for more efficacious treatments and to prevent recurrence and metastasis? While the tumor suppressor role of p53 in breast cancer treatment is well recognized, the evidence supporting an opposite action of p53 in treatment resistance and recurrence in breast cancer is not as clear. Breast cancer recurrence may well be a consequence of the heterogeneous nature of the initial tumor, confounded by the change in hormone signaling from a paracrine to an autocrine proliferative response upon estrogen exposure. Breast cancer cells have a “memory of self”, and investigations in hormone-responsive breast cancer cells after p53 activation show characteristics of aberrant mammary differentiation, followed by dormancy, mediated by p53 activation. Further studies into how p53 protects breast cancer cells from cell death by switching cellular metabolism, downregulating apoptotic proteins, and initiating a cellular differentiation phenotype reminiscent of the normal mammary function is key to our understanding of recurrence in latent breast cancer development and a basis for the design of more efficacious treatments. We need to look to the past to move on to the future and we now have the technology to do so.


CDK2NA protein (p16) immunostaining has been found to be prognostic for human breast cancers, this marker has never been evaluated as a prognostic marker for canine neoplasms. In the present study, the prognostic utility of p53 and p16 was evaluated in 35 canine malignant MGTs. It was observed that 19 (54%) dogs died due to their MGTs with an overall mean survival time (MST) of 882 days. Seven MGTs showed p53 immunostaining, but this was not significantly associated with death  or MST . Five dogs had MGTs with no p16 immunostaining, 28 MGTs had intermediate p16 immunostaining, and two MGTs had increased p16 immunostaining. Neither death due to MGT  nor MST  were significantly associated with p16 immunostaining. Interestingly, p53 immunostaining was significantly associated with an increase or loss of p16 immunostaining. This is the first time that p16 has been evaluated as a prognostic marker for canine neoplasms. While these results suggest that a proportion of canine MGTs develop by cellular mechanisms that alter both p53 and p16 expression, there was no evidence that defects in p53 or p16 alter the behavior of a MGT. Neither p53 nor p16 was found to significantly predict prognosis, although this could reflect the limited number of MGTs included in the study.Mammary gland tumors (MGTs) are common in dogs and show a variable clinical behavior that is difficult to predict. Currently, few immunohistochemical markers have been established to predict the prognosis of a canine MGT. However, p53 immunostaining has been variably reported to be prognostic for canine MGTs. Additionally, while p16 Mammary gland tumors (MGTs) are common in intact female dogs, and 40%–50% of these neoplasms are malignant . The bioOne marker that has previously been evaluated in human and canine MGTs is p53. The p53 protein is considered to be the ‘guardian of the genome’ as it is responsible for detecting DNA damage and triggering apoptosis of damaged cells. Despite the critical role of this protein in cancer biology, studies in humans have typically found only weak associations between prognosis and p53 expression within breast cancers . FurtherCDKN2A protein (p16) is a tumor suppressor protein that prevents cell cycling by regulating the function of the retinoblastoma (pRb) protein [The p16 protein . Cells n protein . Mutatio protein . While l protein ,12. In d protein , althougDue to current uncertainty regarding the prognostic significance of p53 immunostaining for canine MGTs, the ability of p53 immunostaining to predict prognosis was evaluated in a series of canine malignant MGTs. Additionally, the association between p16 immunostaining and prognosis was also investigated in these canine MGTs. This is the first time that p16 immunostaining has been evaluated as a prognostic marker for a canine neoplasm.The databases of two diagnostic laboratories  were searched for cases of canine malignant MGTs for which a diagnostic sample had been submitted for histology. Cases that had been received during 2012–2015 were selected, and surveys were sent to the submitting veterinarians in January 2018. Information requested on the surveys included the treatments that had been used, whether or not the dog had died due to the MGT, and the survival time of the dog. Cases were excluded from the study if the dog had received additional treatments aimed at curing the MGT, although dogs that received palliative treatments, such as anti-inflammatories or antibiotics, were included.Histological sections were evaluated from all cases to confirm the diagnosis of malignant MGTs, and neoplasms were subclassified according to the classification scheme proposed by Goldschmidt et al. .Immunohistochemistry was performed to detect p53 and p16 as previously described ,16. A moCanine MGTs were classified as p53-positive if 20% or more of the cells contained nuclear immunostaining and as p53-negative if less than 20% of the neoplastic cells demonstrated p53 immunostaining. The MGTs were classified as p16-positive, p16-negative, or p16-intermediate. Neoplasms that were p16-positive had intense nuclear and cytoplasmic immunostaining in over 80% of the neoplastic cells. Neoplastic cells within p16-negative MGTs did not contain any significant p16 immunostaining, while p16-intermediate MGTs had low to moderate intensity of p16 immunostaining that was confined to the cytoplasm in over 10% of the cells. The thresholds used to classify the immunostaining for both antibodies were based on the thresholds used most frequently in humans, and more limitedly, in veterinary literature.Differences between groups were investigated by Pearson chi-squared or Fisher’s exact tests and survival times were investigated using Kaplan–Meier and Cox regression analyses using IBM SBSS Statistics v25 .A total of 35 malignant MGTs were included in this study. These included five that were diagnosed in 2012, 12 in 2013, 8 in 2014, and 10 in 2015. The MGTs were subclassified into 8 different histological types. The tumors included 17 simple carcinomas, which were further subclassified into tubular, tubulopapillary, cribriform, and cystic papillary carcinomas .p = 0.73).A total of 28 of 35 (80%) MGTs were p53-negative a, while p = 0.16).Of the 35 canine malignant MGTs, 28 (80%) were p16-intermediate and were characterized by showing a pattern and intensity of immunostaining that was similar to the immunostaining within surrounding non-neoplastic epithelium. In contrast to the p16-positive MGTs, immunostaining in the p16-intermediate MGTs was strictly cytoplasmic. Five (14%) MGTs were p16-negative c, and twp = 0.001).Canine MGTs that were p53 positive were significantly more likely to have altered p16 immunostaining, with only 2 of 7 (29%) p53-positive MGTs being classified as p16-intermediate. In contrast, 26 of 28 (93%) of p53-negative MGTs were classified as p16-intermediate (p = 0.17). Of the dogs with p53-positive MGTs, 4 of 7 (57%) died of their MGTs. This was not significantly different from the dogs with p53-negative MGTs of which 15 of 28  died due to their MGTs. A total of 50% of dogs with p16-intermediate and p16-positive MGTs died due to their MGTs. While 4 of 5 (80%) of dogs with p16-negative MGTs died due to mammary cancer, this difference was not statistically significant (p = 0.46). When MGTs with altered p16 immunostaining (either increased or decreased) were considered as a group, 5 of 7 (71%) of these dogs died due to their MGTs, which was not significantly higher than dogs with p16-intermediate MGTs .A total of 19 (54%) dogs died of their MGTs in this study. Seven dogs died of other causes, and nine dogs were still alive in January 2018. Of the dogs that remained alive at the end of the study, the minimum follow-up time was 807 days after diagnosis. There were no significant differences in survival rates between the different histological types of mammary gland neoplasms (p = 0.57). There were no significant differences in the MST of dogs with p16-positive MGTs , p16-negative MGTs , or p16-intermediate MGTs . When dogs with p16-positive or p16-negative MGTs were considered as a single group, these dogs had a MST of 620 days (95% CI 372–864), which was not significantly shorter than dogs with p16-intermediate MGTs .The overall estimated mean survival time (MST) of the 35 dogs with malignant MGTs in this study was 882 days . Likewise, the MST of the dog with the p16-positive simple carcinoma (131 days) was not significantly different from the MST of the three dogs with p16-negative  or the 9 dogs with p16-intermediate carcinomas .To reduce the potential confounding effect of comparing immunostaining in types of carcinomas that have previously been shown to have different survival times, associations between immunostaining and survival were evaluated in just the 13 dogs with simple carcinomas. In these dogs, the MST of the three dogs with p53-positive simple carcinomas  was not significantly different from the MST of the 10 dogs with p53-negative carcinomas  , and 12 There were no significant differences in the survival rates or the MSTs between dogs with MGTs that were p16-negative, p16-intermediate, or p16-positive. However, a potential role of p16 as a prognostic marker cannot definitively be excluded, as dogs with p16-negative MGTs had the lowest survival rates and there appeared to be a trend for dogs with either p16-negative or p16-positive MGTs to survive for a shorter time than dogs with p16-intermediate MGTs. Similarly, in humans, breast cancers that were p16-positive were reported to have a less favorable prognosis. It is possible that significant differences between the groups were not detected in the present study because of the small number of either p16-negative or p16-positive MGTs that were included. The small number of p16-negative MGTs in the present study (14%) was unexpected, as a previous study had detected loss of p16 immunostaining in 24 of 79 (30%) of canine MGTs . In the In the present study, three distinct patterns of p16 immunostaining were recognized in canine MGTs. The most common pattern consisted of faint to moderate immunostaining that was confined to the cytoplasm. As this p16-immunostaining pattern is similar to that seen in normal epithelium, this staining pattern was considered to represent an intact pRb-p16 pathway. The high proportion of MGTs with this immunostaining pattern suggests that most neoplasms develop without developing mutations within the pRb-p16 pathway. The second pattern was seen in five MGTs and consisted of an absence of p16 immunostaining within the neoplastic cells. This pattern is consistent with the presence of mutations within the p16 gene preventing protein production. Finally, the least common pattern was intense cytoplasmic and nuclear immunostaining in almost all neoplastic cells. Evidence from human oral SCCs suggests that these neoplasms had mutations that prevented the production of pRb . As p16-Canine MGTs with missense mutations in p53 were significantly more likely to also have mutations that disrupted the normal pRb-p16 pathway. It is uncertain why mutations in p53 would be predisposed to additional mutations in pRb or p16; however, the results of the immunostaining in this study suggest that most MGTs develop without missense mutations in p53 or a disrupted pRb-p16 pathway. A smaller subset of MGTs develop as a result of disruption to both pRb-p16 and p53.In the present study of 35 canine malignant MGTs, the histological type was not found to be prognostic. This is in contrast with an earlier study of 229 canine MGTs that revealed a prognostic significance of histological type [As significant differences in survival times between MGTs of different types have been reported , the proFor a prognostic marker to be clinically useful, it has to be shown to consistently predict prognosis when used by multiple different pathologists in multiple different diagnostic laboratories. In veterinary pathology, many prognostic grading schemes and other markers have been proposed, but few have been shown to consistently predict prognosis when widely used . A commoThe results of this study show that canine MGTs demonstrate variable p53 and p16 immunostaining. However, these variations do not appear to influence the biological behavior of the neoplasm sufficiently to significantly alter survival rates or MSTs of the affected dogs. This study is the third to investigate p53 immunostaining as a prognostic marker in canine MGTs and the second to suggest that p53 may not be strongly predictive of prognosis for these neoplasms. This is the first time that p16 has been evaluated as a prognostic marker for canine MGTs.


In recent years, great interest has been devoted to finding alternative sources for human stem cells which can be easily isolated, ideally without raising ethical objections. These stem cells should furthermore have a high proliferation rate and the ability to differentiate into all three germ layers. Amniotic fluid, ordinarily discarded as medical waste, is potentially such a novel source of stem cells, and these amniotic fluid derived stem cells are currently gaining a lot of attention. However, further information will be required about the properties of these cells before they can be used for therapeutic purposes. For example, the risk of tumor formation after cell transplantation needs to be explored. The tumor suppressor protein p53, well known for its activity in controlling Cell Prolif.eration and cell death in differentiated cells, has more recently been found to be also active in amniotic fluid stem cells. In this review, we summarize the major findings about human amniotic fluid stem cells since their discovery, followed by a brief overview of the important role played by p53 in embryonic and adult stem cells. In addition, we explore what is known about p53 in amniotic fluid stem cells to date, and emphasize the need to investigate its role, particularly in the context of cell tumorigenicity. Oct4, Nanog, c-Myc, and Klf4 [Stem cells are present throughout the embryonic development and in adult organs. A coordinated control of stem cell self-renewal and differentiation is essential for maintaining tissue and organ homeostasis. Since 1980, a huge amount of work has been invested into the analysis of embryonic stem (ES) cells. These cells show incredible features like i) the ability to be expanded in vitro while maintaining an undifferentiated state, and ii) pluripotency, which means that the cells possess the capability to differentiate into every cell type of the adult body [and Klf4 . They deand Klf4 . A seconand Klf4 . To overPerinatal stem cells can be derived from postembryonic tissues, which include the tissues sourced at the time of birth, but also comprise the time period from the 16th week of gestation through the neonatal period ,5. TheseThe amnion encloses the amniotic cavity containing the amniotic fluid, a protective and nutrient-containing liquid for the developing fetus . It is mOct4, Klf4, Sox2, Lin28, and c-Myc to induce a pluripotent state, and then differentiated into functional cardiomyocytes using inhibitors of glycogen synthase kinase 3 (GSK3) and Wnt [By using various factors and methods, AFS cells can also be reprogrammed into iPS cells ,24,25. HThe lack of tumorigenesis after transplantation is an interesting feature of AFS cells, although no information is available regarding the underlying mechanisms. Important clues could be gathered by investigating in AFS cells the activities and functions of crucial cell cycle regulators, like the tumor suppressor gene p53.p53 is one of the most well-known and most intensively investigated tumor suppressor proteins. A lot of work has already been done on investigating the role of p53 in ES cells and other adult stem cells and it has been established that apart from its traditional tumor suppressor function, p53 is also reported to be involved in controlling Cell Prolif.eration, self-renewal, and differentiation of stem cells . Amniocentesis is routinely performed between 16–18 weeks of pregnancy. The collected amniotic fluid is used for prenatal genetic testing and as a source of AFS cells. Different approaches and protocols have been used to isolate AFS cells ,31. ThesBased on morphological and growth characteristics, human amniotic fluid cells are grouped into three main categories: i) amniocytes or amniotic fluid specific AF-type cells; this type of cell comprises about 60% of the amniotic fluid cells. ii) Epitheloid E-type cells; this type of cells make about 34% of the amniotic fluid cells. iii) Fibroblastic F-type cells; about 6% of the amniotic fluid cells belong to this type of cells ,40,41. TApproximately 1% of the AFS cells express the surface antigen c-kit CD117), a receptor tyrosine kinase type III, which plays a role in cell survival, proliferation, and differentiation . Some au, a recepAFS cells maintain a normal karyotype and stable telomeres when taken into culture, even after over 200 population doublings , yet othOne of the most important qualities of AFS cells is that they are non-tumorigenic when transplanted into immunocompromised mice . AlthougOct4, Nanog, and Sox2 [Likewise, AFS cells also do not form teratomas after in vivo transplantation . Even 11and Sox2 . Since the discovery of AFS cells, several groups have been exploring their differentiation potential. Even before it was known that the amniotic fluid contains Oct4-positive stem cell-like cells, Streubel and coworkers had demonstrated the expression of skeletal muscle proteins when amniotic fluid cells were cultured in the supernatant of rhabdomyosarcoma cell lines . After tGuan and colleagues showed that undifferentiated human AFS cells express several cardiac genes , acquire a cardiomyocyte-like phenotype after 24-h incubation with 5-aza-2′-deoxycytidine (5-AZA-dC) and develop mechanical and electrical connections with neonatal rat cardiomyocytes when co-cultured . Rat AFSAFS cells have furthermore been demonstrated to form embryoid bodies when cultured in suspension, accompanied by a decrease in the expression of the pluripotency stem cell marker Oct4 and nodal, a marker for the self-renewal potential of ES cells, followed by the induction of differentiation markers like Flk1  for endothelial cells, E-cadherin for epithelial cells, Pax 6 (paired box protein 6) for ectodermal cells, TBXT (T-box transcription factor T) and HBE1 (hemoglobin subunit epsilon 1) for mesodermal cells, and GATA4 for endodermal cells . Similar to ES cells and iPS cells, AFS cells are capable of differentiating into primordial germ cells when cultured under appropriate culture conditions and through formation of embryoid bodies ,71. ThusIn summary, the enormous efforts in the past years by various groups to exploit and investigate differentiation of AFS cells have yielded positive results and demonstrate that amniotic fluid cells have a broad differentiation potential.mdm2 gene and activates its transcription, and the resultant Mdm2 binds to p53, blocks its activity, and mediates the proteolytic degradation of p53 [Functionally, p53 is a transcription factor that elicits its cellular activity mostly through transcriptional activation of target genes. However, besides its primary function as a transcription factor, p53 can also promote apoptosis independent of transcription via direct interaction with pro- and anti-apoptotic proteins . Pertainn of p53 . As a man of p53 , an Mdm2n of p53 . This Mdn of p53 . Addition of p53 . COP1 ann of p53 .p53 is part of a multiple gene family that also includes p63 and p73, as well as several splice variants of the three proteins ,83. p53 The role and effector functions of p53 however are context dependent and such context dependence is affected by cell type, genetic background of the cell, cell microenvironment, and nature of stress the cell is undergoing. Because p53 is such a critical cellular protein, multiple mechanisms have evolved to modulate its activity. The existing strategies consist of manipulating p53’s transcriptional or translational control by altering the level of p53 mRNA, by changing its posttranslational modifications, by altering p53’s half-life by freeing wild type p53 from MDM2, and by altering p53’s intracellular localization . Bax and Puma resulting in apoptosis [Nanog and Oct4 after DNA damage, thus evoking differentiation of these cells [Similar to differentiated cells, p53 becomes activated in stem cells in response to DNA damage, where p53 activates the promoters of its canonical target genes including poptosis . In addise cells ,90,91. BHowever, p53 is not only active in stem cells after DNA damage. A few years ago, it was discovered that p53 is also active in non-stressed stem cells. Yet, the anti-proliferative activity of p53 is compromised in these cells and p53 instead directs a transcriptional program that is highly reminiscent to that of tumor-derived mutant p53 . More recent studies implicate p53 also into the plasticity of stem cells. Here, p53 seems to be involved in the regulation of the self-renewal of stem cells as well as for the onset of differentiation . The ememiR-34a and miR-145. The latter one operates as a direct repressor of the pluripotency factors Oct4, Klf4, Lin28A, and Sox2 upon retinoic acid-mediated differentiation of ES cells [−/− human ES cells further confirmed the suppressive activity of p53 for stem cell pluripotency [The first evidence for a role of p53 in embryos dates back to the 1980’s, when this protein was found highly expressed in primary cell cultures obtained from 12–14 day old mouse embryos but not in 16-day-old mouse embryos . A year ES cells . LikewisES cells . Maimetsipotency ,105,106.ipotency . Silenciipotency . A very ipotency . They shipotency . miRNA-34 family [In adult stem cells including neural and hematopoietic stem cells, p53 negatively regulates proliferation and self-renewal and helps to maintain the quiescent state ,110. Mor4 family . Further4 family . While the role of p53 has been investigated in ES cells and adult stem cells as described above, only little was known about the regulation and function of p53 in AFS cells in the past. Recently, our group investigated p53 protein levels, p53 localization, and p53 activity during Cell Prolif.eration, differentiation, and during the DNA damage response in human AFS cells . Earlier reports showed both constant stable expression and also downregulation of p53 mRNA during long term passaging of AFS cells and AF mesenchymal stromal/stem cells ,113,114.p21 and mdm2 [When we treated AFS cells with retinoic acid to induce neural differentiation, we observed an increase in p53 protein levels  which isand mdm2 . In addiand mdm2 .AFS cells are emerging as an alternative source of stem cells with promising advantages for disease modeling and applications in regenerative medicine, although there are still questions to address regarding their safety and potential, and p53 may play important roles in these processes. As the amniotic fluid contains a heterogeneous population of cells, in many studies, mixtures of unselected, non-clonal cells have been used, causing conflicting results and uncertainty regarding the identity of the cell population employed. However, stem cells within the amniotic fluid do possess a high proliferation rate, clonogenicity, and a broad differentiation potential ranging from multipotent cells to unipotent committed progenitors. The therapeutic potential of AFS cells has been shown in experimental models of diseases of the lung, heart, skeletal muscle, bone, cartilage, kidney, blood and nervous system. Since AFS cells are primary cells of a very early stage of human development, these cells are also interesting as a model system for basic research. Finally, although AFS cells have profiles similar to ES and adult stem cells, it is quite surprising why these cells are not tumorigenic when transplanted into immune-compromised animals. p53, which is already known to be an important tumor suppressor protein in pluripotent stem cells and differentiated cells, may be considered as a crucial control point in AFS cells as well. Therefore, it is essential to identify if p53 is involved not only in the processes underlying the ‘stemness’ of AFS cells but also in their tumorigenicity.p53’s role in tumorigenesis is widely known with thousands of publications and an increasing list of p53 targets and effector functions, making it even more difficult to understand the complexity and reach of this single protein. Interest is also driven to focus on potential mechanistic links between the loss of p53 and the stem cell–like cellular plasticity which has been suggested to contribute to tumor cell heterogeneity and to drive tumor progression. We now know that p53 is actively present and involved in a variety of cellular processes in AFS cells, but its involvement is less well understood. Does p53 play a different cellular function in AFS cells? Is p53 essential for tumor suppressive functions in AFS cells? Does the loss of p53 in AFS cells lead to tumor formation? These are yet unanswered questions, the knowledge of which could potentially improve the usage of AFS cells for therapy in the future.