Thymus extracts or essential oils as natural additives in foods is reduced in comparison with other natural preservative extracts. This review provides an overview of the most important information on the positive effect of the bioactive compounds of thyme and its uses as a preservative in foods, taking into account its origin .Thyme is one of the most important medicinal plants because of its ethnopharmacological relevance and high content of bioactive compounds. This review focuses particularly on thyme as an alternative natural antioxidant and antimicrobial with potential use in the food industry. This is in line with the preferences of the current consumer, who demands healthier and more natural products. Different studies have concluded that the use of thyme increases stability and reduces lipid oxidation during the shelf-life period of foods , which makes thyme a promising source of natural additives. Despite these findings, the use of  Plants produce phytochemicals to protect themselves from bacteria, viruses and fungi, but they also protect food from spoilage when added to food. In recent years, great interest has been focused on using natural preservatives in food products in light of recent studies that have indicated the possible adverse effects related to the consumption of synthetic additives. In addition, natural preservatives improve human health because they protect against diseases .Currently, the natural plant extract industry is moving millions of euros around the world. Approximately 1340 plants are known to be potential sources of antioxidant and antimicrobial components , and morHerbs and spices are among the natural compounds that are currently being used as food preservatives, which contain compounds with marked antioxidant and antimicrobial properties . Plant eMaterial plants, such as herbs of the Labiatae family, have been some of the most studied for their preservative properties . TogetheIn the last decade, a series of investigations have been carried out in order to identify natural substances that are capable of inhibiting lipid autooxidation reactions in meat products and precooked meats after being added during processing operations . SpecialThyme vulgaris, also called common thyme, which belongs to the Lamiaceae family, and it is native to Mediterranean countries in Europe. In addition, Thymus zygis subsp. gracilis grows in southern Spain and northern Africa, although it is mainly represented in Spanish regions, such as east of Andalucia, Albacete and Murcia, and is widely used as a condiment. It is a woody-based herb with aromatic pink flowers and green leaves. This spice is the most commercialized in Spain, where it is distilled to extract its essential oil and also exploited as the “thyme leaf” to obtain natural additives for the food industry. It is highly valuable as a flavoring agent with a spicy taste and commonly added to rabbit, boar and lamb meats. In traditional medicine, thyme has been used as a sedative, a carminative, an additive for baths or an infusion for the treatment of skin diseases [There are several important classes of thyme, such as diseases . From a diseases . HoweverThyme contains monoterpene phenols, including carvacrol, thymol and p-cymene, and other monoterpenes, such as α-pinene, 1,8-cineole, camphor, linalool and borneol .Taking into account that thyme has been extensively studied for its antioxidant and antimicrobial activities with the target of improving the quality of food, the objective of this article is to review the natural preservative properties of thyme and its mechanisms of action against lipid oxidation and microorganisms. In addition, factors that interfere with their forms of application and effectiveness, interactions and synergistic effects are described.Lamiaceae), the genus Thymus  and to the class of Dicotyledons, native to the countries of the western Mediterranean basin. Thyme is one of the medicinal aromatic plants found in the Iberian Peninsula, and its essential oil has become one of the most widely used in the food industry. Thyme has been used since ancient times for its health properties, which are associated with its essential oils and chemical components. Its economic importance is associated with its essential oils.Thyme is an aromatic plant and is widely distributed over the Mediterranean area . Taxonomically, thyme belongs to the family of the Labiatae , thymol  and p-cymene (9.1–22.2%), and other monoterpenes, such as α-pinene (0.9–6.6%), 1,8-cineole (0.2–14.2%), camphor (0–7.3%), linalool (2.2–4.8%) and borneol (0.6–7.5%) .Thymus essential oil is generally a mixture of monoterpenes, which generally contains 10% carvacrol and approximately 50% thymol. The essential oil of thyme is also a source of linalool, α-terpineol, camphor, caryophyllene and γ-terpinene [The composition of erpinene . In addierpinene . Using oerpinene .Of the major compounds in thyme, the content of thymol in Thymus zygis and Thymus vulgaris amounts to 22.3–43.3%and 38.1 %, respectively. In addition, carvacrol accounts for 2.3%, along with other prevailing compounds such as p-cymene (29.1%), g-terpinene (5.2%) and linalool (3.7%) . The cheIt is important to consider the main factors that influence the effectiveness of natural extracts and essential oils; for example, the biotic characteristics of the plants, such as the season and geographical source, have an influence on the concentration and composition of the bioactive compounds . In addiThymus sipyleus, and they reported different constituents in their essential oil since, in one of them, mostly alcohols such as borneol (11.2%) were identified, while the other variety was characterized by the presence of carvacrol (58.1%) and thymol (20.5%). The authors concluded that phenolic oils were more effective in all tests performed.In the Labiatae family, thymol is always accompanied by its carvacrol isomer, and both are constituents of the most studied thyme essential oil in terms of its antioxidant activity. Yanishlieva et al.  comparedIn general, the inhibitory capacity of oxidation presented by thyme extracts is fundamentally due to its richness in phenolic compounds . The antThymus zygis and determined that there is an apparent relationship between the antioxidant potential of these extracts and the total phenols that they contain. One of the sources of the antioxidant properties of the phenols in thyme is their ability to exhibit redox properties and neutralize free radicals [Phenolic constituents appear to be primarily responsible for the antioxidant properties of these substances. However, Dorman et al.  establisradicals .Thymus riatarum showed a high DPPH radical-scavenging capacity and ferric reducing power in ethanolic, aqueous and methanolic extracts. In addition, Golkar et al. [The antioxidant activity of thyme has been demonstrated through several in vitro and in vivo methods. Using in vitro methods, Aouam et al.  reportedr et al.  showed tThymus vulgaris. These authors identified a new component, p-cimen-2,3-diol, isolated for the first time from thyme leaves, which showed higher antioxidant activity than that of α-tocopherol and BHA (butylhydroxyanisole). Moreover, Youdim et al. [Thymus zygis and its major components, confirming that the effectiveness of these substances increased in the following order:In a comparison of the antioxidant activity between thyme and other antioxidant compounds, thyme was reported to be one of the best antioxidants. Focusing first on the mechanism of the antioxidant activity of thyme, Lee and Shibamoto  conductem et al.  carried Essential oil > thymol > carvacrol > Υ-terpinene > mircene > linalool > ρ-cimene > limonene > 1,8-cineool > α-pinene.According to these ranks, all the substances tested have antioxidant activity, although, as can be seen from the work of the abovementioned authors, no single component is more effective than the essential oil as a whole.The antioxidant effect is due to the fact that thyme EO contains at least 60 bioactive compounds with powerful antioxidant properties. The main phenolic compounds are carvacrol (3.5%), thymol (68.1%), ρ-cimeno monoterpene hydrocarbons (11.2%) and γ-terpinen (4.8%) , which hThymus vulgaris extract as a protector against the oxidation of the lipids present in biological membranes, identifying components in the extract with substantial antioxidant power. Moreover, flavonoids that have been isolated from the concentrate of this plant show comparable activity to that of BHT, α-tocopherol or L-ascorbic acid [The antioxidant effect of thyme extract has been analyzed in various studies, such as that carried out by Haraguchi et al. , who verbic acid . In the bic acid , it was Gavaric et al.  reportedFurthermore, Nagoor Meeran et al.  studied In a study that compared thymol with other terpenes, such as limonene, menthone, linalool, caryophyllene, camphor, caryophyllene, menthol and pulegone, Salgado-Garciglia et al.  reportedThymus vulgaris) helps these animals retain a favorable antioxidant capacity throughout their lives [Using in vivo methods, it has been proven, for example, that supplementing the diet of rats with thyme essential oil . These kinds of diets are related to lower incidence rates of chronic diseases, including certain forms of cardiovascular disease and cancer, and these effects are due to the synergistic and the additive effects of the complex mixture of the bioactive compounds contained in fruits, vegetables, spices and herbs. Western societies tend to focus on a single food instead of whole dishes. A diet rich in spices not only enhances the taste of food but also makes it healthier.Infusions of thyme have been used for their health benefits for thousands of years as a cold treatment, antispasmodic, a carminative agent, traditional medicine in digestive problems and an expectorant for upper respiratory tract infections. In addition, the potential therapeutic effects of thymol have been used for the treatment of disorders affecting different organ systems, such as the cardiovascular, nervous, and respiratory systems ,84,85.Thyme vulgaris at 100 mg/kg/day for 8 weeks significantly reduced blood pressure in rats, which exhibited improvements in hypertension and aortic vascular damage. Moreover, Haque et al. [In general, phenolic compounds have an influence on cardiovascular disease because they reduce molecular damage, prevent plaque formation and inhibit the oxidation of cholesterol. Several researchers have also reported the beneficial health effects of thyme on cardiovascular disease. Kensara et al.  reportede et al.  studied e et al. . Moreovee et al. ,88,89.Thymol, which contributes to the antioxidant activity of thyme, is considered an important antioxidant with therapeutic properties. In general, the bioactive components of spices that have shown potent cancer-preventive, anti-inflammatory, anti-atherogenic and antimutagenic bioactivities are in fact antioxidants, which is mainly because these phenolic compounds have a beneficial role in preventing diseases mediated by oxidative stress. For example, several studies have suggested that thymol inhibits lipid peroxidation  and the The ability of thyme to act as an anti-inflammatory agent has been reported in several in vitro and in vivo studies. This herb improved the activity of the enzyme superoxide dismutase (which acts as an anti-inflammatory agent) . MoreoveThymus spp. with concentrations of thymol ranging from 10.05 to 71.15%, and these authors concluded that thymol reduced the gene expression of tumor necrosis factor-alpha, IL-1B and IL-6 in macrophages.Ocaña and Reglero  studied Thymol also plays a role in the antimicrobial activity of thyme EO and has been reported to show an antiseptic property that is 30 times higher than that of other phenol compounds. These components show antifungal and antibacterial bioactivity against both pathogenic microflora and food spoilage microflora.Erwinia amylovora, such as the study of Karami-Osboo [Colletotrichum gloeosporioides and Rhizopus stolonifera [Thymus sp. showed strong antifungal activity against the pathogens Penicillium italicum, Penicillium digitatum and Botrytis cinerea [C. albicans and A. niger. The main compound contained in the oil with anti-fungicidal activity is camphor.Other studies have shown the antimicrobial properties of thyme essential oil against the Gram-negative bacterium mi-Osboo . Moreovelonifera . Thymus  cinerea . In this cinerea  reportedS. epidermidis, E. coli, Micrococcus mirabilis, B. subtilis, S. typhimurium, Enterobacter cloacae, S. aureus, S. enteritidis and P. aeruginosa [Thymus vulgaris extract has also shown antibacterial activity against ruginosa .Since ancient times, herbs have been used to improve the look and taste of food; however, herbs and spices can also decrease the use of other unhealthy ingredients, such as synthetic additives , sugar, fat and salt.The use of thyme in food is limited almost entirely to meat products, where it is used for technological purposes, mainly as an antioxidant and preservative. The literature published so far demonstrates that the direct addition of natural antioxidants from thyme can inhibit the development of rancidity in different types of foods. In this context, numerous studies have been carried out that have reported the preservative properties of thyme.The features of the food (to which thyme is added) can influence the bioactivity of compounds and their preservative properties in foods; such factors include storage conditions, composition and the types of microorganisms in the food. All of these aspects are related to their preservative properties . In addiThe proximate composition of food is very important, and several interactions have been described between protein and phenolic compounds that reduce the action of the bioactive compound; similarly, fat content was reported to have a protective effect on bacteria . MoreoveThe main objective of the use of thyme in food is to extend the shelf life; however, the main limiting aspect for the use of the essential oil and plant extract of thyme is the development of negative organoleptic characteristics in foods, contributing to an unpleasant odor and taste. To avoid these sensory limitations, several strategies can be employed, such as the use of low concentrations and other conservation methods or the inclusion of natural compounds encapsulated with nanocarriers or added to bioactive films.On the one hand, the essential oil can be encapsulated with nanocarriers, such as nanoemulsions, nanofibers, cyclodextrins or amylose , which mOn the other hand, natural compounds can be included in bioactive films in order to increase the sensory acceptability while also allowing the gradual release of the compound and avoiding the negative organoleptic effects .In spite of the demonstrated potential of thyme and its constituents in vitro, its use as a food preservative has been limited by the organoleptic problem. In many food products, hydrophobic EO compounds are impaired by several interactions with the components of the food matrix, such as fat ,106, staGiven these interactions between natural extracts and food components, it is useful to study how the constituents of the EO can interact with the food matrix. This interaction can be studied by measuring the microorganism’s growth in a culture medium containing different concentrations of starch, protein and fat.Different studies have been carried out on thyme to study its properties for stabilizing lipids in meat and meat products. Harpaz et al.  reportedThymus vulgaris) into diet rats contributed to the maintenance of an excellent antioxidant capacity in these animals [To explore the effects of the inclusion of thyme in the animal diet, a study with distilled thyme leaf was conducted by Moñino et al. , who inv animals . Moreove animals  studied w/w.As previously stated, the beneficial effects of the addition of thyme, both its extract and its essential oil, have been studied in several types of meat: veal , lamb 1, chickenp < 0.05) at the level of 0.9%. Therefore, the administered level affected the acceptability. Along these same lines, previous researchers have reported that high levels of essential oils (which are necessary to achieve bacteriostatic activity against pathogens in food) would not be applicable to food because they result in negative organoleptic properties [Along the same lines, Ouattara et al.  showed toperties ,124.Other studies have investigated whether the addition of natural extract containing phenolic compounds or inclusion of rosemary or thyme leaves in the diet of pregnant ewes has an influence on the oxidative stability and sensory properties of raw and cooked lamb, poultry, as well as pork and chicken meat ,135,136.Another form of thyme application is the use of its by-products, such as distilled thyme leaves. These by-products are generated because the thyme essential oil industry generates an excess of waste (distilled thyme leaves) after the distillation of the leaves for the extraction of EO. The effect of supplementation on animal diets with thyme by-products on meat quality was studied by Nieto et al. , who invA. hydrophila and L. monocytogenes in cooked and chilled chicken meat. These authors concluded that thyme extract could be useful as an antimicrobial in chicken meat.To study the antimicrobial effect of thyme on meat, Hao et al.  studied Thymus spathulifolius essential oil on 15 species of molds and yeasts, and 9 of the 15 were inhibited. These results coincide with those shown by Lambert et al. [L. monocytogenes in pork sausages. Ozcan et al. [Escherichia coli O157:H7, while at a concentration of 0.2%, it showed higher antimicrobial activity. In addition, Sagdic et al. [Escherichia coli O157:H7 that was dependent on the dose.Sokmen et al.  demonstrt et al. , Burt [1t et al.  and Hollt et al. . For thet et al.  showed tn et al.  reportedc et al.  showed tListeria monocytogenes [Listeria monocytogenes in minced pork [Escherichia coli, A. hydrophila, Yersinia enterocolitica and Salmonella pullorum. Among the oils studied, thyme showed strong antimicrobial activity against A. hydrophila. Thymol reduced yeast growth because it caused damage to metabolic and structural enzymes and inhibited the repair mechanisms of heat damage in yeasts [Thymus has been reported by numerous studies [Shigella has been thoroughly shown by Bagamboula et al. [As previously stated, thymol and carvacrol have been shown to possess in vitro antimicrobial activity against Gram-positive bacteria ,140,141.ytogenes . Specificed pork . In addiced pork  evaluaten yeasts . The ant studies ,146,147. studies , and itsa et al. .Escherichia coli has been shown in vitro in several studies [Escherichia coli or Salmonella in minced beef [The effect of thymol and carvacrol against  studies ,150,151.ced beef  or in prced beef . The effced beef  in threeced beef . This beced beef , who stuced beef  observedced beef ,160,161.ced beef , could bced beef , which, ced beef . Carvacrced beef ,164. TheThe mechanism of inhibition of thyme EO has not been studied in great detail , but somS. sonnei and S. flexneri, showed that carvacrol, followed by thymol, demonstrated the greatest antibacterial activity against all microorganisms tested; however, linalool and estragol showed a limited effect.The antimicrobial activity of thyme EO has been studied in vitro in model food systems ,124 and The strategy of using natural extracts as fish and seafood preservative agents is a response to the consumer’s concern about synthetic additives and environmental impacts . In genePseudomonas spp. and Enterobacteriaceae.To explore the effects of polymer films containing thyme essential oil, Jouki et al.  studied Pseudomonas fluorescens and Pseudomonas aeruginosa; however, in accord with these results, Huang et al. [Pseudomonas spp.Moreover, Gómez-Estaca et al.  studied g et al.  showed tVibrio parahaemolyticus. For example, Yano et al. [Vibrio parahaemolyticus was weak at low temperatures.Another food-borne pathogen related to the intake of lightly cooked or raw seafood is o et al.  (2006) rTo investigate sensory acceptability, Navarro-Segura et al.  studied In addition, several examples of the use of thyme in fish have been reported, such as the study of Mahmoud et al. , who shoMentha piperita L.), chamomile (Matricaria chamomilla L.), nettle (Urtica dioica L.), yarrow (Achillea millefolium L.) and thyme (Thymus vulgaris L.), and they achieved positive results in milk yield and the physicochemical and technological composition of milk. In general, the success of the use of antioxidants in the diet of animals has been confirmed by many authors as a tool to improve the quality of products and derivatives, and their benefits for animal health, although the administration of these antioxidants must be controlled in terms of the quantity and the type of antioxidant administered since frequent use of phenolic compounds in the diet of cows can cause abortion or intoxication [The effects of supplementing animal diets with thyme derivatives and their influence on milk quality were reported by Kraszewski et al. . These axication .T. zygis leaves into goat feed resulted in cheeses and milk with added bromatological values, which increased the oxidative stability of a typical cheese with wine [The antioxidant activity of thyme is especially useful in food preservation. For example, the incorporation of ith wine . In thisThe European Commission has accepted different EO components as flavoring in food, such as thymol, eugenol, carvacrol, citral, vanillin, limonene, linalool, carvone and cinnamaldehyde, because they do not present a risk to consumer health. The FDA (United States Food and Drug Administration) classifies these substances as GRAS . Included in this category  are thyme, clove, cinnamon, oregano, mustard, nutmeg and basil. In addition, in the USA, before an EO can be added to foods, there are regulatory limitations on the accepted daily intake; therefore, before adding an EO, a daily intake survey should be available for evaluation by FDA.In general, recommendations for the intake of food for healthy eating do not yet include suggested amounts of spices and herbs. Therefore, the recommended intake of spices and herbs should be considered for incorporation into guides for healthy eating in different countries. In addition to the health effects, the use of this plant in foods can be used to totally or partially replace other less desirable ingredients, such as synthetic additives, sugar or salt. The addition of these herbs and spices may be a strategy to stabilize stir-fry dishes, dressings and marinades, casseroles, curries, soups and Mediterranean-style cooking.Before the application of thyme as a natural extract, several factors must be taken into account, such as the nature of the extracts, fruiting stage, mode of extraction, the concentration of active extract components and the possible synergistic effect between thyme and other components. Thyme extract can be applied as a complementary food preservative in food systems such as milk products, dressings, meat and oils and for the enhancement of functional foods. In addition, other considerations regarding thyme are important and warrant its inclusion in the food industry, such as its non-toxicity, availability and low cost.Thyme has been extensively studied for its antioxidant and antimicrobial activities. Currently, new advances and techniques in food technology have facilitated efficient identification, processing and extraction of bioactive compounds from herbs and spices in order to include them in functional foods and nutritional supplements. The strategy to produce bioactive compounds with decreased negative organoleptic characteristics and enhanced shelf life will increase overall applications in foods and will subsequently increase spice consumption and, as a consequence, have a positive impact on human health.However, just a few essential oils or plant extracts containing phenolic compounds are currently included in foods. These essential oils and natural extracts represent potential replacements of competitive synthetic antioxidants and antimicrobials in food and possible value-added products for human consumption. Nevertheless, there is much to learn in terms of its stability in specific matrices and the relationship between their structures, the biological activity of the bioactive metabolites, synergistic effects and effective doses. These themes are the subject of current and future research. Although more studies are needed to establish the broad spectrum of the health benefits of thyme, recent results are very encouraging. Greater public awareness of the properties and benefits of spices to human health would increase the intake of these functional food products.In conclusion, the development of functional foods enhanced by the inclusion of thyme with value-added properties is of great interest to the scientific community and to the food industry. Thus, beyond their role in flavor, spices should be considered natural components of our nutrition when added to food.


Donor‐specific anti‐human leukocyte antigen (HLA) antibodies (DSAs) in recipients is a risk factor for donor stem cell graft failure in haploidentical hematopoietic stem cell transplantation (haplo‐HSCT), and the treatment to reduce the levels of DSAs is not unanimous. This study was to analysis the role of DSAs for stem cell engraftment and to discuss the effective treatment to reduce DSAs in haplo‐HSCT.We retrospectively evaluated the levels of DSAs and the effect of the combination treatment of rituximab and donor platelets (PLTs) for donor stem cell engraftment in haplo‐HSCT patients from June 2016 to March 2018 at our center.11) before donor stem cell infusion and the other three patients received a combination therapy of donor apheresis platelets and rituximab due to the antibodies against both donor HLA‐I antigens and HLA class II (HLA‐II) antigens. All the seven patients achieved donor stem cell engraftment successfully, and the DSAs levels decreased rapidly after transplantation.Nine patients (11.5%) out of the total 78 patients were DSAs‐positive and multivariate analysis revealed DSAs was the only factor that affected engraftment. Seven out of the 9 DSAs (+) patients received therapy: Four had antibodies against donor HLA class I (HLA‐I) antigens and were administered two therapeutic amounts of donor apheresis platelets (platelet count approximately 3‐5 × 10DSAs is an important factor affecting engraftment in haplo‐HSCT. Donor platelet transfusion is one simple and effective treatment for HLA‐I DSAs, and a combination therapy should be administered if patients have both HLA‐I and HLA‐II antibodies. However, the probability of finding human leukocyte antigen (HLA) matched sibling donors is <30%, and it is even lower in unrelated donors.22.1Consecutive patients (N = 78) who received a haplo‐HSCT at our Stem Cell Transplant Center between June 2016 and March 2018 were enrolled in the study. The HLA‐A, HLA‐B, HLA‐C, HLA‐DQB1, and HLA‐DRB1 were sequenced in both directions using SeCore SBT Kits (One Lambda). The products were separated on ABI 3730 Genetic Analyzer (Hitachi High‐Technologies Corporation) and analyzed using uTYPE6.0 analysis software (One Lambda). In the present study, haplo‐HSCT was defined as HSCT from donors who share one of the two HLA haplotypes, regardless of the number of HLA‐mismatched loci in A, B, C, DQ, and DR. The study was approved by the ethics committees of the Institute of Hematology, Chinese Academy of Medical Science & Peking Union Medical College according to the guidelines of the Declaration of Helsinki. All patients provided informed consent before participating in the study.2.2Serum samples were collected from the patients for screening anti‐HLA antibodies with a LABScreen panel reactive antibody (PRA) Kit (One Lambda). Positive samples were further tested for the specificity of the antibodies against HLA‐I  and HLA‐II  antigens using a LABScreen Single Antigen Kit (One Lambda). Fluorescence was measured using a Luminex100 flow analyzer (Luminex), and the data were analyzed using the LABScan 100 software (One Lambda). The median fluorescence intensity (MFI) of the PRA beads’ reactions was obtained from the output file generated by the flow analyzer, adjusted for the background signal using the formula: sample beads −negative control beads. The fluorescence intensity of the negative and positive control beads was <100 and >9000, respectively. If the sample data did not fit these conditions, the serum or plasma was treated with ADSORB OUT beads (One Lambda) to reduce background fluorescence. All samples with fluorescence intensity >500 were tested with single Ag beads to confirm and identify sample specificity. MFI was adjusted for the background signal using the abovementioned formula. The samples were considered: negative, MFI < 500; weakly positive, MFI 500‐2000; positive, MFI 2000‐10 000; and strongly positive, MFI > 10 000.2.32); cytarabine (6 g/m2); and rabbit antithymocyte globulin  or pig ATG (80 mg/kg). In patients with aplastic anemia (AA), the conditioning included CTX 150 mg/kg; Flu 120 mg/m2; and rabbit ATG 12.5 mg/kg. Cyclosporin A or tacrolimus plus short‐term methotrexate along with mycophenolate mofetil were used for aGVHD prevention. Donor stem cells  were obtained according to the opinions of the donors .Myeloablative conditioning regimens for patients with hematologic malignancies were as follows: intravenous busulfan (Bu 9.6 mg/kg) or total body irradiation (10 Gy separated by 3 days); cyclophosphamide ; fludarabine  was additionally used 1‐2 weeks before the conditioning(14‐21 days before donor stem cell infusion) combined with two therapeutic amounts of donor apheresis PLT infusion on the day −1 (1 day before donor stem cell infusion). For patients who had DSAs against donor HLA‐I antigens without antibodies against donor HLA‐II antigens, only two therapeutic amounts of donor apheresis PLT infusion were administered on day −1 to reduce the levels of DSAs.For patients who had DSAs against both donor HLA‐I antigens and HLA‐II antigens, one dose of rituximab  chimerism analysis. BM puncture was performed on 14, 28, 42, and 60 days after donor stem cell infusion. Chimerism analysis involved the quantitative polymerase chain reaction (PCR) of informative STRs in the recipient and donor. DNA was amplified with fluorescent PCR primers for markers that would distinguish the donor and recipient alleles. Fluorescent PCR products were separated using an Applied Biosystems 3730 Genetic Analyzer (Applied Biosystems), and the Genemapper software (Applied Biosystems) was used to correlate allele peak areas with the percentage of donor or recipient DNA. It was defined as complete donor chimeras when the result of STR was ≥97%. The ratio of X/Y chromosomes was tested using fluorescence in situ hybridization if the gender of donor and recipient was not matched.Neutrophil recovery was defined as an absolute neutrophil count (ANC) >0.5 × 102.5P < .05.SPSS 21.0 was used for data processing and analysis. Fisher's exact test was used to compare the differences of engraftment rate among groups, whereas multivariate analysis was performed with logistic regression analysis to identify risk factors that affect donor cell engraftment. Statistical significance was considered when 33.1Overall, 78 patients who underwent haplo‐HSCT from June 2016 to March 2018 were included in this study, and 69 patients were DSAs (−) and 9 (11.5%) were DSAs (+). The characteristic of the patients was showed in Table P = .001). Multivariate analysis revealed that DSAs was the only factor that affected engraftment . The age, HLA mismatch, gender of the donors, ABO mismatch, disease status, stem cell source and amount were all irrelevant to PGF patients (Patients 1‐4) underwent routine myeloablative conditioning, which was the same as the other 69 DSAs (−) patients, and no special treatments for DSAs were additional used before donor stem cell infusion. PGF occurred in three patients . The engraftment rate of the 4 DSAs (+) patients  was much lower than that of DSAs (−) recipients  in this study including the first four patients (Patients 1‐4) mentioned above and the following five new DSAs (+) patients Patients 5‐9). The characteristics were summarized in Table ‐9. The c2) was additionally used 1‐2 week before conditioning (14‐21 days before donor stem cell infusion) and two therapeutic amounts of donor apheresis PLTs (PLT count was approximately 3‐5 × 1011) were infused on −1 day  had antibodies against donor HLA‐I antigens without antibodies against to donor HLA‐II antigens. Only two therapeutic amounts of donor apheresis PLT infusion were additionally adopted at day −1 to reduce the levels of DSAs for the 4 patients, and all of them obtained donor cell engraftment  had DSAs against donor HLA‐I and HLA‐II antigens, so combination treatments were used: one dose of rituximab 375 mg/m mg/m2 wa3.3The median fluorescence intensity (MFI) was used semiquantitatively for estimation of levels of DSAs and for predicting crossmatch results and assessing immunological risk. Crossmatch assays, the patient's serum was incubated with the donor's T and B lymphocytes to assess antibody reactivity directly, were not done in this study. The DSAs levels of Patient 1 and Patient 2 in their secondary transplants and Patients 5‐9 in their primary transplants were shown in Table 4AnasettiIt was difficult to speculate the exact DSAs levels due to the limited DSAs (+) patients in our study. In ChangMethods used to decrease the total antibody levels were reported as follows: plasmapheresis or immunoabsorption for antibody removal, rituximab or proteasome inhibitor for inhibition of antibody production, intravenous immunoglobulin (IVIg) or donor HLA antigens simulant for antibody neutralization, and monoclonal antibodies for inhibition of complement cascade.In our study, total seven patients (two patients for their secondary haplo‐HSCT and five patients for their first haplo‐HSCT) received therapy to lower DSAs levels during transplants and they all obtained complete donor cell engraftments. Patient 1, Patient 7, and Patient 9 had both anti‐HLA‐I and anti‐HLA‐II DSAs, so Rituximab was administered once 1‐2 weeks before the conditioning regimen to inhibit B lymphocytes producing new antibody and donor PLTs used as donor HLA‐I antigens simulant was infused at −1 day to neutralize existing DSAs before donor stem cell infusion. Patients 2, Patient 5, Patient 6, and Patient 8 possessed DSAs against donor HLA‐I antigens; hence, only donor PLT infusions was adopted to lower anti‐HLA‐I DSAs. The results showed that the DSAs levels decreased to a very low level at 1 month and could not be tested at 2 months after transplantation. We speculated that the myeloablative conditioning could also inhibit donor lymphocytes producing new antibodies and donor mononuclear cells infused at 0 day also consumed recipients’ DSAs partly.Donor PLT infusion was the only special treatment for four patients with anti‐HLA‐I DSAs meanwhile without anti‐HLA‐II DSAs in this study because HLA‐I antigens are highly expressed on PLTs and donor PLTs could act as donor HLA‐I antigens simulant to neutralize the recipients’ anti‐HLA‐I antibodies. The levels of anti‐HLA‐I DSAs decreased rapidly without increasing the risk of developing acute GVHD, and all the four patients obtained complete donor cell engraftments. Other studies showed the similar results.In our study, plasmapheresis was not used due to: (a) most of our patients were diagnosed as high‐risk MDS without remission and the peripheral blood platelet counts were very low before conditioning, the risk of serious bleeding would be increased during or after plasmapheresis; (b) the day before donor stem cell infusion such as −2 or −3 days was preferred for plasma exchange but there were two risk factors: Bleeding because of lower count of blood platelets after conditioning and ATG concentration decreased.Most of the DSAs (+) patients in our research were diagnosed as MDS, and they underwent multiple blood products administering before stem cell transplant, which could be one of the causes of producing PRA/DSAs. CiureaIn conclusion, PRA/DSAs is an important factor that may affect donor cell engraftment in haplo‐HSCT. Routine PRA/DSAs testing should be incorporated in donor selection prior to transplantation, and DSAs‐positive donors should be avoided. If there is no other alternative suitable donor, it is recommended that appropriate therapy should be taken to improve donor stem cell engraftment. Donor PLT transfusion is a simple and effective treatment to neutralize anti‐HLA‐I antibodies without the risk of increasing GVHD and other adverse effect, and a combination treatment should be administered if the patient has both anti‐HLA‐I and anti‐HLA‐II antibodies.


Citri Sarcodactylis Fructus (CSF) is widely used as folk medicine in traditional Chinese medicine (TCM). The dried and steam-processed CSF (SCSF) has been employed for harmonizing the stomach over thousands of years under the guidelines of TCM theory. However, little is known about the differences in chemical compositions between CSF and SCSF. Moreover, the gastroprotective effects of CSF and SCSF on ethanol-induced gastric mucosal injuries in rats have yet to be investigated. Consequently, the present study aimed to investigate the chemical differences and gastroprotective effects of CSF and SCSF, providing some experimental framework for the development of CSF and SCSF.  The chemical compositions of CSF and SCSF extracts were determined using an ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometer (UPLC-Q-TOF-MS), and their gastroprotective effects of different doses were assessed in rats with ethanol-induced gastric injuries on the levels of oxidative stress and inflammatory cytokines.  A total of 42 components were identified in CSF and SCSF, and most of them were flavonoids, limonoids, coumarins, and glycosides. There were no differences in the compositions between CSF and SCSF, but the relative contents of the components were different. Among them, nine screened compounds were considered as potential discriminating markers responsible for the differences between CSF and SCSF. Besides, pretreatments with CSF and SCSF markedly improved the gastric mucosal injuries in rats for their antioxidant and anti-inflammatory properties. And SCSF exhibited a better gastroprotective effect than CSF.  The compositions of CSF were unchanged after steam-processing, while the relative contents of their components were changed. These changes may be the major reasons for the differentiation of their efficacies. In addition, CSF and SCSF could alleviate ethanol-induced gastric mucosal injury through the enhancement of antioxidant and anti-inflammatory activities. SCSF exhibited a better gastroprotective effect than CSF, which emphasized the necessity of steam processing. Citrus medica L. var. sarcodactylis Swingle. It is officially named as Fo Shou in China. It has been used in traditional and folk medicines for thousands of years to treat liver-qi stagnation, chest pain, stomach irritation, loss of appetite, vomiting, frequent cough, and excessive phlegm, by harmonizing stomach, relieving the depressed liver, and reducing dampness and phlegm . However, it has been reported that the crude CSF possesses the property of dryness, which is prone to deplete qi and injure yin as a result of long-term administration. Processing is capable of altering the chemical constituents of the Chinese medicinal material, which can change its TCM property for treating different syndromes +. Therefore, UPLC-Q-TOF-MS was operated in both positive and negative ion modes to obtain more comprehensive information. The peak areas were changed between CSF and SCSF. The content of glycosides was decreased in SCSF compared to that in CSF, which may be attributed to the decomposition of glycosides during steam processing. Compared with CSF, the changes of limonoids, with higher nomilinic acid, obacunone, and limonin contents and lower nomilin content in SCSF, indicate that limonoids may be converted internally. In addition, saccharides and amino acids were reduced after steam processing, suggesting that the Maillard reaction may occur during this process. Furthermore, compounds 16 and 38 appeared at m/z 192.04 Da, while compounds 17 and 31 appeared at m/z 202.02 Da, with the same molecular weight. Their MS/MS fragment ions were almost the same because they had similar structures but different retention times. Therefore, we speculated that these compounds might be isomers, and further investigation is needed to distinguish them.According to our results of chemical composition analysis, both CSF and SCSF were mainly composed of flavonoids, limonoids, and coumarins. The flavonoids tended to generate [M − H]The chemical information of CSF and SCSF were analyzed by the OPLS-DA model to identify and distinguish their variabilities. In OPLS-DA analysis, the VIP value is the most commonly used index to evaluate the contribution of variables, and it is generally considered that the variables with a VIP > 1 are deemed statistically significant . HoweverThe gastric injury caused by alcoholism is a problem that raised worldwide concern. Alcohol would primarily destruct the gastric mucosa resulting in the depletion of mucus and bicarbonate accompanied by the enhancement of oxidative stress and inflammation followed by gastric injury , 11. To Ethanol-induced gastric injury is often accompanied by the production of reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide and hydroxyl radical, which in turn leads to cell death by interacting with a large number of molecular factors. Both enzymatic and nonenzymatic antioxidants, such as SOD and GSH, can scavenge these oxygen free radicals and protect against gastric tissue damage . Howeverα, a potent stimulator of neutrophil infiltration, can activate and accumulate neutrophils around the injury sites, resulting in gastric microcirculatory disturbance and gastric damage. In addition, TNF-α can promote the secretion of interleukin-1, IL-6, and other cytokines to trigger inflammation [α, IL-1β, and IL-6 in various types of tissues [α, IL-2, and IL-6, indicating their anti-inflammatory effects against ethanol-induced gastric injury. A distinct impact of the model group was observed for IL-10 production in gastric tissue, in which the levels of IL-10 in SCSF groups were almost normalized to basal level as compared to the normal group. Although unexpected, these results are consistent with those reported in another research [Cytokine networks contain a variety of cell damaging factors that lead to the development of gastric injury. The increased levels of several proinflammatory cytokines were observed in gastric tissue after ethanol administration. TNF-ammation . IL-2 plammation . IL-6 isammation . IL-10 i tissues . In the research . It is nBecause of the complex composition of CSF and SCSF, further investigations are needed to comprehensively attribute the observed differences in pharmacological effects to individual or several compounds.Our findings indicated that there are no differences in chemical compositions between CSF and SCSF, but the contents of their compounds are different. In addition, the gastroprotective effects of CSF and SCSF against ethanol-induced gastric mucosal injury could be attributed to their antioxidant and anti-inflammatory activities, in which SCSF exhibit better gastroprotective efficacy than CSF. These experimental results encourage further studies on CSF and SCSF.


Alzheimer’s disease (AD) is the major cause of death in adults with Down syndrome (DS). There is an urgent need for objective markers of AD in the DS population to improve early diagnosis and monitor disease progression. NPTX2 has recently emerged as a promising cerebrospinal fluid (CSF) biomarker of Alzheimer-related inhibitory circuit dysfunction in sporadic AD patients. The objective of this study was to evaluate NPTX2 in the CSF of adults with DS and to explore the relationship of NPTX2 to CSF levels of the PV interneuron receptor, GluA4, and existing AD biomarkers (CSF and neuroimaging).n = 49), prodromal AD  and AD dementia . Non-trisomic controls (n = 34) and patients with sporadic AD dementia  were included for comparison. We compared group differences in CSF NPTX2 according to clinical diagnosis and degree of intellectual disability. We determined the relationship of CSF NPTX2 levels to age, cognitive performance , CSF levels of a PV-interneuron marker (GluA4) and core AD biomarkers; CSF Aβ1–42, CSF t-tau, cortical atrophy (magnetic resonance imaging) and glucose metabolism ([18F]-fluorodeoxyglucose positron emission tomography).This is a cross-sectional, retrospective study of adults with DS with asymptomatic AD , pDS  and dDS . This reduction was similar to that observed in sporadic AD . CSF NPTX2 levels were not associated with age (p = 0.6), intellectual disability (p = 0.7) or cognitive performance . Low CSF NPTX2 levels were associated with low GluA4 in all clinical groups; controls , adults with DS  and sporadic AD . In adults with DS, low CSF NPTX2 levels were associated with low CSF Aβ1–42 , low CSF t-tau , increased cortical atrophy (p < 0.05) and reduced glucose metabolism (p < 0.05).Compared to controls, mean CSF NPTX2 levels were lower in DS at all AD stages; aDS (0.6-fold, adj.Low levels of CSF NPTX2, a protein implicated in inhibitory circuit function, is common to sporadic and genetic forms of AD. CSF NPTX2 represents a promising CSF surrogate marker of early AD-related changes in adults with DS. Down syndrome (DS) has been conceptualized by the International Working Group for Alzheimer’s disease (AD) as a genetically determined form of AD  due to tAccumulating evidence suggests that early dysfunction of inhibitory interneuronal circuits contributes to cognitive abnormalities in AD decades before clinical disease onset . It folln = 94). We have included non-trisomic population controls (n = 34) and sporadic AD patients (n = 40) from the Sant Pau Initiative for Neurodegeneration (SPIN) cohort for comparison.Based on the aforementioned data, we hypothesized that, as a genetic form of AD, DS CSF will have reduced NPTX2 and correlated GluA4 levels, reflecting PV interneuron dysfunction, thus representing a potential surrogate marker of early AD pathophysiology in adults with DS. To test this hypothesis, we have evaluated CSF levels of NPTX2 as a surrogate marker of inhibitory circuit dysfunction using the Down Alzheimer Barcelona Neuroimaging Initiative (DABNI) cohort, the largest collection worldwide of CSF and neuroimaging data collected from adults with DS , determined according to our internal cut-offs -FDG. After computerized tomography data was obtained, brain PET was acquired. The reconstruction method was iterative  with a 128 × 128 image size, 2 mm pixel size and 2 mm pixel slice thickness. [18F]-FDG-PET images were intensity-scaled by the reference pons-vermis region -fluorodeoxyglucose (FDG) positron emission tomography (PET) scan on an integrated PET-TC system (Philips Gemini TF). The breakdown of diagnosis in these individuals was as follows: aDS s region , spatials region  and thens region . With ths region . All resr2 values were adjusted for the number of predictors in the model. Where residuals deviated from a Gaussian distribution (Shapiro-Wilk p < 0.05), square root transformed values were used. Outliers were excluded using the 3 × IQR rule. For the neuroimaging analyses we performed Monte-Carlo simulation with 10,000 repeats (family wise error [FWE] correction at p < 0.05). Due to the small size of each clinical group with available neuroimaging, analyses were performed on the complete DS dataset only. The figures show only those results that survived FWE correction. Alpha was set at 0.05 for global analyses and 0.01 for post-hoc analyses.Statistical analyses were performed in R version 3.4.3 . Group dn = 168). Compared to controls the mean age was higher in the sporadic AD group , lower in the aDS group  and comparable in pDS and dDS (both adj.p > 0.9). As would be expected for an age-related disease such as AD, mean age was lower in aDS than pDS  and dDS . The proportion of males:females included in the study was 46:54 and was comparable across diagnostic groups (p = 0.09). The proportion of individuals with severe/profound intellectual disability was comparable across AD stages (p = 0.7). All cognitive measures were lower in sAD compared to controls  and in dDS compared to aDS . CSF biomarker levels were as previously reported [1–42 levels were lower in all groups  and p-tau and t-tau levels were higher in sAD, pDS and dDS .The Table reported . Brieflyn = 168, p < 0.0001). Specifically, post hoc analyses showed that compared to controls, mean NPTX2 levels were 0.5-fold in sporadic AD , 0.6-fold in aDS , 0.5-fold in pDS  and 0.3-fold in dDS . Mean NPTX2 levels were comparable between sporadic AD and DS  and across AD stages in DS .Mean CSF NPTX2 levels were associated with clinical diagnosis  and were not associated with any measure of cognitive performance in DS . Mean NPTX2 levels were not associated with age overall (p = 0.6) or at any AD stage .In adults with DS, CSF NPTX2 levels were not associated with level of intellectual disability , low NPTX2 levels correlated with low GluA4 levels in controls , sAD  and in DS : The association in DS was apparent at all AD stages; aDS , pDS  and dDS .We next sought to determine whether the PV-interneuron specific marker, GluA4, shows a coordinated reduction with NPTX2 in the CSF. While CSF GluA4 levels were not associated with clinical diagnosis , pDS  and dDS . Low NPTX2 levels were also associated with p-tau , pDS  and dDS . Similar correlations were observed for CSF t-tau .We next sought to determine the relationship between CSF NPTX2 levels and core CSF AD biomarkers in DS. Low NPTX2 levels were associated with low CSF Aβels Fig. a in all top; n = 63, FWE < 0.05) and with reduced glucose metabolism in more widespread regions of the temporal and parietal cortices .We also analyzed the relationship between CSF NPTX2 levels and structural and functional brain changes in adults with DS. As shown in Fig. Neuronal Pentraxin Receptor 2 (NPTX2) has recently been identified as a novel CSF biomarker of inhibitory circuit dysfunction in sporadic AD , 15. Her1–42 or amyloid PET tracers) and tau-mediated neurodegeneration (CSF p-tau and t-tau) combined with neuropsychological assessment, which can be confounded by substantial inter-individual variation in intellectual disability. NPTX2 represents a new addition to the DS biomarker arsenal that may be used to detect early signs of AD in this relatively under-studied population. Furthermore, this work opens the door to future studies exploring the potential of CSF NPTX2 as a prognostic marker for late-onset myoclonic epilepsy in Down syndrome and sporadic AD.DS cohorts with available CSF are scarce and current biofluid markers for AD in the DS population are restricted to surrogate markers of amyloidosis (CSF AβThe lack of association of NPTX2 levels with intellectual disability suggests that the reduction in NPTX2 levels is not neurodevelopmental. This is supported by the finding that reduced CSF NPTX2 levels were associated with increased cortical atrophy and reduced glucose metabolism in temporal and parietal regions of the brain, regions that are particularly susceptible to AD pathology. Thus, CSF NPTX2 levels inversely correlated with the extent of both neuronal loss and neuronal dysfunction in signature AD regions. Taken together, we propose that changes in CSF NPTX2 levels in adults with DS are related to AD pathophysiology and not to intellectual disability.1–42, a surrogate CSF marker of increased aggregation of brain Aβ1–42. This is the first evidence that a synergized increase in brain Aβ1–42 aggregates and reduced NPTX2 can be measured indirectly in the CSF of adults with DS. NPTX2 is expressed by pyramidal neurons and secreted by axon terminals to mediate activity-dependent strengthening of pyramidal neuron excitatory synapses on GABA-ergic PV interneurons, a mechanism that is dependent upon the AMPA receptor subunit, GluA4 [Low CSF NPTX2 levels in adults with DS were associated with low CSF GluA4 and low CSF Aβt, GluA4 . Histolot, GluA4 , regionst, GluA4 . This ist, GluA4 . Studiest, GluA4 –39 have t, GluA4 .Thus, a Unlike NPTX2, GluA4 levels were not significantly reduced in CSF of adults with DS compared to controls, making NPTX2 a potentially more valuable CSF biomarker in DS. Based on these findings, CSF NPTX2 can be added to the growing list of biomarkers that show changes similar, both in direction and magnitude, to those described in sporadic or autosomal dominant AD , 40. The1–42, t-tau and p-tau are excellent diagnostic markers in sporadic AD and in the DS population and we do not expect NPTX2 to improve upon those markers. Rather, this study supports the increasing literature that suggests CSF NPTX2 as a surrogate marker of underlying interneuronal circuit dysfunction in sporadic and, now genetic, forms of AD even at the preclinical stage. Further studies in longitudinal cohorts are needed to explore the full potential of CSF NPTX2. The capacity of NPTX2 as an early prognostic marker would be one potential use worth exploring for example. Alternatively, as tau and Aβ are common drug targets in AD clinical trials, a surrogate measure of synaptic dysfunction that does not directly measure the drug target levels would be a useful addition to the biomarker arsenal.CSF AβIn previous studies in sporadic AD, CSF NPTX2 levels correlated better with cognitive performance than the core AD biomarkers . FurtherThe association of increased CSF NPTX2 levels with increased CSF p-tau levels in the DS population could indicate that widespread neuronal loss at later disease stages may lead to increased NPTX2 clearance into the CSF and may mask a reduction in NPTX2 synapses. Nevertheless, the inverse relationship we report between CSF NPTX2 levels and a neuroimaging marker of cortical atrophy, suggests that neurodegeneration has a discernable but limited confounding effect on CSF NPTX2 levels.A limitation of this study is the cross-sectional design; longitudinal studies are needed to fully establish the prognostic value of NPTX2 in both sporadic AD and in DS. Furthermore, these findings warrant replication in an independent DS cohort. Exploration of the potential of NPTX2 as a blood biomarker would also be valuable to minimize the use of lumbar puncture in people with DS.Here we report that CSF NPTX2 levels are reduced in adults with DS compared to cognitively normal, non-trisomic controls even prior to AD onset. Similar reductions were observed in the CSF of sporadic AD patients and have been previously reported in postmortem AD and DS brains , 15. Sub


Histiocytic sarcoma affecting the central nervous system (CNS HS) in dogs may present as primary or disseminated disease, often characterized by inflammation. Prognosis is poor, and imaging differentiation from other CNS tumors can be problematic.To characterize the clinicopathological inflammatory features, breed predisposition, and survival in dogs with CNS HS.One hundred two dogs with HS, 62 dogs with meningioma.Retrospective case series. Records were reviewed for results of cerebrospinal fluid (CSF) analysis, CBC, treatment, and outcome data.P ≤ .001). Corgis and Shetland Sheepdogs had predominantly primary tumors; Rottweilers had exclusively disseminated tumors. Marked CSF inflammation was characteristic of primary rather than disseminated HS, and neoplastic cells were detected in CSF of 52% of affected dogs. Increased neutrophil to lymphocyte ratios were seen in all groups relative to controls (P <.008) but not among tumor subtypes. Definitive versus palliative treatment resulted in improved survival times (P < .001), but overall prognosis was poor.Predisposition for CNS HS was seen in Bernese Mountain Dogs, Golden Retrievers, Rottweilers, Corgis, and Shetland Sheepdogs (Clinicopathological differences between primary and disseminated HS suggest that tumor biological behavior and origin may be different. Corgis and Shetland Sheepdogs are predisposed to primary CNS HS, characterized by inflammatory CSF. High total nucleated cell count and the presence of neoplastic cells support the use of CSF analysis as a valuable diagnostic test. Prognosis for CNS HS is poor, but further evaluation of inflammatory mechanisms may provide novel therapeutic opportunities. APCantigen presenting cellCIconfidence intervalCNScentral nervous systemCSFcerebrospinal fluidHShistiocytic sarcomaIQRinterquartile rangeMSTmedian survival timeN:Lneutrophil to lymphocyte ratioRBCred blood cellTNCCtotal nucleated cell countTPtotal protein concentration1Histiocytic sarcomas (HS) are tumors that arise typically from interstitial dendritic cellsThe majority of published data about CNS HS relates to primary, localized CNS HS in small case series,To further evaluate the inflammatory nature of CNS HS in dogs, a clinicopathological review of a series of primary and disseminated CNS HS cases and meningiomas was done. Markers of inflammation in CSF (TNCC) and peripheral blood (leukocyte neutrophil to lymphocyte [N:L] ratio) were defined in the context of tumor subtype  to determine potential relevance to diagnostic and prognostic criteria.2This was a retrospective investigational study conducted at the University of California (UC) Davis School of Veterinary Medicine and Department of Small Animal Clinical Sciences, Virginia Maryland College of Veterinary Medicine. Medical records were reviewed for cases with a primary inclusion criterion of a histopathological diagnosis of HS affecting the CNS. Definitive classification as primary CNS  or disseminated (CNS disease with evidence of other organ system involvement) required full necropsy including the CNS. Cases were classified as brain or spinal cord based on primary neuroanatomical localization at presentation. Additional clinical and clinicopathological data were collected including signalment, clinical history, CSF analysis, CBC, treatment history, and survival.2.1Diagnosis of HS was made by a board‐certified pathologist (R.J.H. or K.W.) based on previously described histopathological and immunophenotypic features.2.2Overrepresentation of HS in specific breeds was determined only for dogs diagnosed with CNS HS (n = 91) admitted to the UC Davis Veterinary Medical Teaching Hospital (VMTH) compared to the total UC Davis VMTH reference population (n = 222 814) during the study period (1986‐2018).2.3Cerebrospinal fluid analyses were included from cases in which samples were acquired at first presentation, before specific treatment. The CSF collection site was noted when available. Any corticosteroid administration within 1 week of CSF analysis was noted. Total nucleated cell counts, total protein concentration (TP), RBC counts, and differential counts were performed within 30 minutes of collection of CSF as previously described.2.4Inclusion criteria for N:L ratios were the same as for CSF analysis. Complete blood counts from cases that had received corticosteroids within 1 week of presentation were excluded from analysis. Only VMTH cases were included, and reference values for N:L ratios were calculated from raw CBC reference interval data from the UC Davis VMTH clinical laboratory.2.5Treatment groups were defined as definitive if specific treatment modalities including surgical resection, radiation treatment, or chemotherapy  were used in any combination, and palliative if anti‐inflammatory , corticosteroid, antiepileptic, or other analgesic medications were the only therapeutic interventions. Survival was defined for all cases from time of presentation with neurological signs, and only for cases with survival times >1 day to exclude animals euthanized at the time of imaging or diagnosis.2.6To define breed predisposition, a Fisher's exact test was used to compare the distribution of HS within each breed relative to the entire population of dogs admitted to the UC Davis VMTH during the retrospective period, and the corresponding odds ratios were presented with surrounding 95% confidence intervals (CI).P < .05 was considered statistically significant.Continuous variables were assessed for normal distribution using a combined test of skewness and kurtosis. Rates of steroid administration were compared between groups using Fisher's exact test. Cerebrospinal fluid, TP, TNCC, and CBC N:L ratios were compared according to steroid administration status within the overall population and within histiocytic and meningioma subgroups using Wilcoxon rank sum tests. Among all dogs, and in the subset with no corticosteroid administration, CSF TP, CSF TNCC, and CBC N:L ratios were compared across tumor subgroups  using Dunn's test of multiple comparisons using rank sums. Overall survival time measured from clinical presentation was estimated using the Kaplan‐Meier product limit method. Dogs alive or lost to follow‐up at the time of data collection were censored in analyses. Rates of definitive versus palliative treatment were compared between primary and disseminated HS cases using Fisher's exact test. Survival estimates were compared according to treatment type  overall and within primary and disseminated HS groups. All tests were 2‐sided, and 33.1Between 1986 and 2018, 102 dogs were identified with a histopathological diagnosis of CNS HS . Tumors Breed predisposition was determined from 91 cases presented to the UC Davis VMTH with a control hospital population of 222 814 dogs during the study period. Seven breeds had ≥3 CNS HS . Two tumors also had spinal cord involvement. Based on MR imaging, involvement of both meninges and brain parenchyma was a consistent finding for intracranial HS and most tumors had broad areas of meningeal contact. All intraventricular tumors were found in animals with multifocal disease, and 1 disseminated tumor involved only cranial nerves III, IV, and V with no parenchymal involvement.For HS affecting the spinal cord, 7/8 primary HSs were intrinsic compared to disseminated HS where 24/25 tumors were extradural. Vertebral bone involvement was documented in 14/25 of the disseminated tumors with extradural lesions. No apparent regional predisposition was seen with primary HS , but disseminated HS was most common in the thoracic region . One primary and 2 disseminated tumors also involved brain.3.2P = .58) between HS  and meningioma  cases.Cerebrospinal fluid analysis was available for 24 primary CNS HS  and 17 disseminated CNS HS  cases Figures ; Data S3P = .12, P = .67, respectively). Median CSF TNCC differed significantly (P < .001) across tumor types  and 62 dogs with meningioma . No significant difference (P = .09) was found between the disseminated HS and meningioma groups.Median CSF TNCC did not differ significantly based on the presence or absence of corticosteroid administration for both HS and meningiomas groups (P = .02) between HS cases that received steroids  and HS cases that did not . Median TP of CSF did not differ significantly (P = .75) for meningioma cases based on corticosteroid administration. For all dogs, CSF TP differed significantly (P < .001) across tumor types  and disseminated HS . No significant difference (P = .12) was found between primary and disseminated HS groups. Significance conclusions remained unchanged when CSF TP concentration analysis was restricted to cases with no corticosteroid administration .Median CSF TP differed significantly P = .0 between Neoplastic cells were identified in 52%  of cytological preparations for which slides were available for review Figure . Neoplas3.3P = .08) across tumor types . Median N:L ratio was 4.2  in 12 dogs with primary HS, 8.6  in 21 dogs with disseminated HS, and 7.7  in 42 dogs with meningioma. Median N:L ratio in 60 control dogs  was significantly lower than in the primary HS (P = .007), disseminated HS (P < .001), and meningioma groups  longer survival compared with palliative treatment   and definitively vs palliatively treated disseminated HS .Survival time after presentation for neurological signs was available for 96 of 102 cases . Ninety‐four dogs died and 2 were lost to follow‐up. Treatment modalities and survival data are outlined in 4Our study defines hospital population‐based breed predispositions for HS affecting the CNS in dogs and defines several clinicopathological characteristics that suggest primary and disseminated HS may have different pathophysiology in the context of CNS disease. Primary and disseminated CNS HS were shown to differ in terms of inflammatory response based on CSF analysis, different breed predisposition, and different anatomical locations of disease, particularly with disease affecting the spinal cord.The presence of breed predisposition for HS affecting the CNS likely represents underlying genetic susceptibilities, but disease‐related factors may be complex, reflecting not only susceptibility to HS in general but also to development of primary versus disseminated CNS disease. This complexity is apparent in our data in which larger populations of primary and disseminated CNS HS were analyzed. Corgis have been reported to have a high frequency of HS affecting tissues outside the CNSThe underlying cell of origin may be different for primary versus disseminated CNS HS. The term histiocyte covers a spectrum of cell types including dendritic antigen presenting cells (APC) or those of macrophage origin.The previously reported predisposition in Labrador Retrievers was not confirmed in our study, but the suspected predisposition in Shetland Sheepdogs as proposed previouslyPrimary CNS HS was associated with an inflammatory phenotype compared to disseminated CNS HS (and meningioma) based on CSF TNCC. We can speculate that the specific cell lineage of primary versus disseminated disease is associated with a more pro‐inflammatory phenotype, but other factors may be associated with anatomical localization of the neoplastic process. Primary HS spinal cord tumors were predominantly intrinsic compared to extradural disseminated tumors, and a logical argument can be made that these primary tumors would be more likely to stimulate an inflammatory response, reflected in CSF pleocytosis within the subarachnoid space. However, other intrinsic and intradural spinal cord tumors generally are not associated with the marked pleocytosis seen with primary CNS HS,Systemic inflammatory responses associated with cancer are well documented, and increases in peripheral blood N:L ratios have been used widely as an indicator of tumor‐associated inflammation.Limited data are available regarding treatment and survival for dogs with CNS HS, but anecdotal reports suggest prognosis is generally poor. Case series of CNS HS with >3 dogs have reported MST of 3 days (n = 19),Our study conclusions are limited by relatively small sample sizes in tumor and treatment subgroups, variability in defined survival end points and potential for inappropriate categorization of primary tumors, even with comprehensive necropsy examination. However, the significant differences defined between primary and disseminated CNS HS particularly in terms of breed predisposition and inflammatory response within the CNS are notable. Advances in treatment of a variety of cancers in humans by manipulation of inflammatory and associated immune‐cell modulation have resulted in new optimism about several previously intractable cancers. Primary CNS HS in dogs appears to carry a poor prognosis regardless of standard therapeutic interventions. Further evaluation of the inflammatory environment present in these tumors may provide critical insight into the biology of CNS HS in dogs and the potential for the development of novel therapeutic approaches.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.Authors declare no IACUC or other approval was needed.Authors declare human ethics approval was not needed for this study.Appendix S1: Supplementary MaterialClick here for additional data file.Appendix S2: Supplementary MaterialClick here for additional data file.Appendix S3: Supplementary MaterialClick here for additional data file.


Alzheimer’s disease (AD) is a multifaceted neuronal disorder and a challenge to medical practitioners, as the blood–brain barrier (BBB) acts as a major obstacle for drug delivery to the brain. Development of a nanomaterial-based drug delivery system (DDS) paved a way to penetrate the BBB. Starch, a ubiquitous natural biopolymer, has received much attention as a DDS due to its biocompatibility, biodegradability and eco-friendly nature. The present study focuses on encapsulating methyl gallate (MG) within starch nanoparticles (starch-encapsulated MG (SEMG)) and assesses its neuroprotective potential against β-amyloid (Aβ)-induced toxicity, the key factor for AD pathogenesis in Neuro2A cells. SEMG showed potent acetylcholinesterase inhibitory, antioxidant activity and anti-amyloidogenic activity by attenuating the fibrillation of Aβ and destabilizing the preformed mature fibrils. Furthermore, SEMG also attenuated the cytotoxic effect induced by Aβ in Neuro2A cells (50% inhibitory concentration 18.25 ± 0.025 μg/mL) by mitigating reactive oxygen species (ROS)-mediated macromolecular damage, restoring mitochondrial membrane potential and attenuating apoptosis. Characterization of SEMG revealed amorphous rock-shaped structure with average particle size of 264.6 nm, exhibiting 83% loading efficiency and sustained release of drug, with 73% release within 24 h at physiological pH. Overall, the outcome of the present study signifies starch as a promising nanocarrier for the delivery of drugs for the treatment of AD. Starch is an inexpensive renewable polysaccharide ubiquitously found in nature with wide application in industries. Multiple evidences revealed the use of this biopolymer as an encapsulating agent to preserve compounds such as vitamins, lipids, drugs, microorganisms and flavoring agents, polyphenols and herbicides from adverse conditions such as light, temperature and pH, etc. . MesoporTerminalia myriocarpa, Terminalia chebula, Terminalia arjuna Bergenia ciliata (hairy Bergenia) Geranium niveum and Paeonia anomala s—methyl gallate in the supernatant.[MG]Release kinetics of MG were assessed under in vitro condition based on the methodology of Winarti et al. . ApproxiThe swelling nature of MG-loaded starch nanoparticle was evaluated by suspending pre-weighted dried MG-loaded starch nanoparticles in 10 mL of phosphate-buffered saline (pH 7.4) at 37 °C. Weight of the swollen starch nanoparticle was determined at various time intervals. The swelling ratio was calculated based on the following equation:−1 with a resolution of 4 cm−1. Morphology of SEMG was examined using atomic force microscopy (AFM) using NT-MDT  and zeta potential (ZP) analyzer .Absorption spectrum of SEMG was assessed using a UV-Visible spectrophotometer  by scanning between 200 and 800 nm with resolution of 1 nm. Crystalline nature of SEMG and MG alone was characterized by X-ray diffraction using the X’ Pert PRO Analytical X-ray diffractometer with Cu Kα radiation, operating at a voltage of 40 kV and current of 30 mA . FTIR (Fourier-transform infrared spectroscopy) spectra of SEMG and MG alone were analyzed using the FTIR-Nicolet Thermo spectrophotometer iS5 (USA) instrument by scanning between 4000 and 400 cmFree radical scavenging activity of SEMG and MG was assessed by the DPPH scavenging assay based on the methodology of Shimada et al. . Differe3, and the absorbance was measured at 700 nm.Reducing power of SEMG was evaluated based on the methodology of Oyaizu . SEMG inAcetylcholinesterase (AChE) inhibitory activity was assessed based on the methodology of Ingkaninan et al. , with slAβ (25–35), purchased as lyophilized powder, was dissolved in 1,1,1,3,3,3,hexafluoro 2 propanol (HFIP), sonicated for 2 h. Reaction mixture was lyophilized, reconstituted in 1 mL milli-Q water to a final concentration of 1 mM and stored at –20 °C until further use.Kinetic study on effect of SEMG against Aβ aggregation was evaluated by treating freshly prepared solution of Aβ (25–35) monomers (100 μM) in Tris-HCl (pH 7.4) in the presence and absence of various doses of SEMG (25 and 50 μg/mL) for 96 h at 37°C. Every 6 h, aliquots of reaction mixture were taken and assessed for aggregation based on the Thioflavin-T (Th-T) assay.Freshly prepared solution of Aβ (25–35) monomers in Tris-HCl (pH 7.4) to final concentration of 100 µM was incubated at 37 °C for 20 h to form oligomers. SEMG (50 µg/mL) was incubated with oligomeric mixture for 48 h and aliquots were drawn from the incubation mixture at 20 and 48 h respectively, for spectrofluorimetric and confocal microscopy analysis by Th-T assay based on the methodology of Khurana et al. . AliquotMature fibrils were formed by incubating Aβ (25–35) in Tris-HCl (pH 7.4) for 96 h. Ability of SEMG to destabilize the mature fibrils was assessed by incubating SEMG (50 μg/mL) for 9 days and the aliquots were drawn for spectroscopic and confocal microscopic studies.2 incubator at 37 °C.Neuro2A cells obtained from the National Center for Cell Sciences (NCCS), Pune, India, were used as an in vitro model system to assess the β-amyloid (Aβ)-induced toxicity. Cells were cultured in DMEM medium containing 10% FBS and 1× penicillin-streptomycin in a CO5 cells/mL) were treated with various doses of Aβ (25–35) (10–50 µM) for 24 h followed by PBS wash and treatment with MTT (1 mg/mL) for 3 h at 37 °C. The cells were subjected to PBS wash and the blue-colored formazan crystals were solubilized by DMSO (Dimethyl sulfoxide). Absorbance was measured at 540 nm using a multi-plate reader. Protective effect of SEMG (20–100 μg/mL) was assessed by pre-treating with SEMG for 2 h prior to Aβ (25–35) at 20 μM concentration and incubated for 24 h, and then subjected to the MTT assay [Pre-seeded Neuro2A cells (2 × 10TT assay .50) (18.25 ± 0.025 µg/mL) for 2 h followed by Aβ (20 µM) for 24 h. Cells were harvested and treated with DCFH-DA (10 µM) for 30 min at 37 °C in the dark followed by PBS wash to remove the extracellular DCFH-DA, and examined under a fluorescent microscope . Quantification of intracellular ROS was carried out by incubating the cells in lysis buffer  and the intensity of fluorescence was measured using a multi-mode plate reader with the excitation/emission wavelength of 485/535 nm, respectively.Intracellular reactive oxygen species (ROS) level was evaluated by measuring the change in fluorescence as a result of oxidation of the fluorescent probe DCFH-DA  . Neuro2ALoss of ΔΨm in cells treated with SEMG was analyzed using lipophilic cationic dye (JC-1), based on the methodology of Sivandzade et al. . Cell liLipid peroxidation was assessed according to the method of Ohkawa et al.  and the v/v) to a final concentration of 10 µg/mL for 10 min. The cells were visualized under a fluorescence microscope (excitation and emission 490/525 nm). Each field of cells was photographed (magnification ×400) for calculation of the relative fluorescence intensity. The cells with condensed or fragmented nuclei were counted as apoptotic cells. All experiments were repeated thrice and about 100 stained cells were counted in 10 randomly selected fields.Acridine orange and ethidium bromide (AO/EtBr) dual-staining technique was used to visualize nuclear changes and apoptotic body formation of the characteristic features of apoptosis . Followip < 0.05) comparing between the groups control vs treated. IC50 values were calculated using Probit software .All the experiments were conducted in triplicate and one-way analysis of variance (ANOVA) (SPSS 17) was used to compare the mean values of each treatment. Significant differences between the means of parameters were determined by using the Duncan’s test  were shown in  gallate . On the articles . HoweverThe two-dimensional (2D) micrograph of AFM displays the randomly arranged nanoparticles of SEMG without any kind of agglomeration, and the three-dimensional (3D) topography of AFM shows the possible protrudes of SEMG A. MorphoDLS analysis revealed that the average particle size of SEMG was observed to be 264.6 nm B based oIn vitro drug release kinetics of SEMG were assessed in PBS  at physiological pH 7.4 for 24 h under constant stirring. The surface erosion and swelling behavior of nano-carrier (SEMG) upon the PBS system initiates the process of breaking of hydrogen bonds between the starch nanoparticles and MG, which is significantly responsible for the release of MG . ConcentAlzheimer’s disease is the acquired dementia characterized by the progressive degeneration of neurons, leading to cognitive deficits and behavioral symptoms, which intensify with time, creating a major health challenge for the medical practitioners . Current50 value of SEMG and MG was observed to be 15.25 ± 0.024 and 35.25 ± 0.04 µg/mL, respectively. Results illustrate that starch encapsulation restored the AChE activity of MG, indicating it as a suitable drug delivery system.In AD, the major causative agent for cognitive decline is degeneration of cholinergic neurons associated with loss of neurotransmission due to reduction in the level of neurotransmitter acetyl choline (ACh) . As curr50 values of nano-encapsulated drug, drug alone and standard BHT were observed to be 9.83 ± 0.05, 26.05 ± 0.025 and 12.04 ± 0.04 μg/mL, respectively.Mounting evidences revealed that oxidative stress observed in early stages of AD plays a vital role in triggering complex signaling pathways, leading to pathogenesis of AD and lesion formation . AntioxiReducing capacity of compound depends on the electron-donating ability, which was assessed based on the ability to convert ferricyanide complex to ferrous form, and the change in color was measured at 700 nm. Aberrant misfolding and aggregation of Aβ peptide into fibrils is the critical event which aggravates the pathogenesis of AD, hence researchers focused on investigating compounds that avert aggregation of Aβ peptide. Several phyto-compounds rich in polyphenolic content have been reported to attenuate the aggregation of Aβ peptide or destabilize the preformed fibrils. In addition, the drug MG has been reported for its potent anti-aggregation and Aβ mature fibril destabilizing ability . The preThe SEMG (50 and 100 μg/mL)-treated group showed a decrease in fluorescence intensity in the elongation phase, illustrating minimal Aβ fibrillation. In addition, SEMG (100 μg/mL)-treated groups showed increased lag phase of aggregation, revealing the fact that SEMG might have stabilized the native unfolded peptide, thereby intervening the aggregation of Aβ peptide. In a Phase I study, Aβ (25–35) peptide showed a five- and eight-fold increase in fluorescent intensity at 20 and 48 h, revealing the formation of protofibrils and oligomers from Aβ monomer. Upon co-treatment with SEMG, the fluorescent intensity reduced by four-fold (4.15 ± 0.019 a.u.) and ten-fold (2.25 ± 0.03 a.u.) after 20 and 48 h, illustrating the fact that SEMG efficiently attenuated the formation of oligomers from Aβ monomers B. ResultIn a Phase II study, an increase in fluorescence intensity was observed in Aβ (25–35)-treated groups from 96 h (30.89 ± 0.63 a.u.) to 9 days (50.22 ± 0.24 a.u.), revealing the formation of mature plaques from oligomers. Treatment with SEMG and galanthamine showed a remarkable decline in fluorescent intensity by four- and six-fold when compared to vehicle control, indicating the disaggregation of mature fibrils and attenuation of self-assembly of mature fibrils to amyloid plaques by SEMG A. Furthe50 value of SEMG was observed to be 18.25 ± 0.025 µg/mL (The neuroprotective effect of SEMG against Aβ (25–35)-induced toxicity was assessed by the MTT assay. Results revealed that treatment of Neuro2A with Aβ (25–35) for 24 h reduced the viability of the cells dose-dependently, with a reduction by 49.72% ± 4.27% at the highest dose of 50 µM, and this dose was fixed for further studies A. Pretre25 µg/mL B. The in25 µg/mL . Phase c25 µg/mL C.2O2 in the presence of transition metal ions Fe2+ or Cu2+, which in turn promotes Fenton chemistry enhancing the production of ROS and RNS, leading to lipid peroxidation and protein oxidation [Multiple evidences revealed that Aβ peptide during oligomerization induced the release of Hxidation ,43. In tAβ (25–35)-treated cells showed a significant increase in nitric oxide level (0.729 ± 0.018 mM of nitrate/mg of protein), while SEMG-treated groups reduced the nitric oxide level (0.213 ± 0.008 mM of nitrate/mg of protein) similar to vehicle control (0.249 ± 0.019 mM of nitrate/mg of protein), which might be due to the antioxidative potential of SEMG C.Multiple laboratory and clinical evidences revealed the presence of elevated levels of oxidative stress markers of lipid peroxidation and protein oxidation, like 4-hydroxynonenal and protein carbonyl content, in the brain of AD patients ,45. To vThe changes in MMP induced by Aβ toxicity were assessed by using lipophilic fluorescent dye JC-1. Fluorescent microscopic studies revealed that vehicle control cells showed uniformly red fluorescent cells (JC-1 aggregates), indicating the presence of intact mitochondrial transmembrane, while Aβ-treated cells showed loss of red fluorescent cells complemented with appearance of green fluorescence cells (JC-1 monomer), revealing the fact that dye diffused into cytoplasm due to disruption of MMP—the initial step of the apoptotic pathway C. HoweveLoss of MMP due to Aβ toxicity leads to activation of the apoptotic pathway, leading to neuronal death. In the present study, the anti-apoptotic effect of SEMG against Aβ-induced toxicity was assessed by the AO/EtBr dual-staining technique, widely used to identify morphological changes associated with apoptosis. Results showed the presence of uniformly stained green fluorescence cells, illustrating viable cells in vehicle control, while Aβ-treated cells showed the presence of orange red fluorescent cells, indicating late apoptotic stage B. SEMG cTo assess the genotoxic effect of Aβ (25–35) in Neuro2A cells, single-cell gel electrophoresis was carried out. Fluorescence microscopic images showed the presence of large non-fragmented intact nucleoids in vehicle control-treated cells, while Aβ-treated cells showed an increase in the length of DNA tail (DNA damage), which provided the appearance of comet during alkaline gel electrophoresis C. SEMG-tThe present study reported, for the first time, fabrication of starch-encapsulated methyl gallate by the graft polymerization technique. Fabricated SEMG showed high encapsulation efficiency and sustained release of drug MG under physiological pH 7.4. SEMG exhibited acetylcholinesterase inhibitory activity and antioxidant capacity. In addition, SEMG also attenuated aggregation of Aβ peptide and disaggregated the preformed amyloid plaques, illustrating the fact that SEMG restored the anti-amyloidogenic effect of MG. SEMG exhibited neuroprotective effect by attenuating ROS-mediated mitochondrial dysfunction and DNA damage induced by Aβ toxicity in Neuro2A cells. To conclude, the results of the present study revealed that starch-based nano-carriers are suitable for the delivery of the drug methyl gallate for the treatment of Alzheimer’s disease and other related neurodegenerative disorders.