Furthermore, the results

Furthermore, the results Selleck AC220 from the individual chromatograms (Fig. 1B) of the venoms clearly demonstrates the presence of isoforms of crotamine, crotapotin and phospholipase A2, of the crotoxin complex. The phospholipase A2 isoforms were found in more abundance, being observed in the majority of the chromatograms of the studied groups. Despite the

high variability in the concentrations, the chromatographic profiles did not present variation of venom proteins when considering the captivity time and ontogenetic variation. The RP-HPLC profile of the crotamine-positive and -negative venoms can be observed in Fig. 2A and B respectively. The contents of major peaks were determined by manual collection of the fraction followed by Edman degradation. In the case of crotapotin, the peptide was reduced with Dithiothreitol and alkylated with iodoacetamide (following standard protocols) and resubmitted to another chromatographic separation before individual sequencing of the peaks. The results on toxicity and coagulant activity are presented in Table 2 that shows LD50 statistical difference between males and females wild life groups. Statistical difference was observed also for clotting BIBF 1120 mouse time (CT) to newborns and reference venom group. The group of animals

inoculated with crotamine-positive venom presented hypertonicity of the hind paws followed by complete paralysis. The groups inoculated with crotamine-negative venom and control did not present any neurological activities. The monitoring of biological, biochemical and pharmacological activities of venoms should be one of the great concerns of institutions that produce antivenom, given that studies conducted in the last 50 years have demonstrated variation in these activities attributable

to sex, Linifanib (ABT-869) age range, geographic origin, diet, captivity, season of the year and/or possible environmental changes (Ferreira et al., 2009, 2010a, in press; Campagner et al., in press; Schenberg, 1959b; Furtado et al., 2003; Pimenta et al., 2007; Calvete et al., 2009). This variability has direct implications on the antivenom type produced and, depending on the favorable response or lack thereof, on the treatment of snakebite patients (Chippaux et al., 1991; Warrel, 1997; Calvete et al., 2009). In the present study a large number of adult Cdt snakes (males and females) and newborns were evaluated, comparing the biological activities of animals newly born in the wild with those maintained for at least three years in captivity, finding a high variability in their venoms. Thus, the protein profile evaluated did not present a difference among the adult individuals corroborating Cárdenas et al. (1995) who found values of 76.9% in Brazilian snakes and 81.4% for Argentinean ones. On the other hand, the venom from the offspring showed a protein content of 60%, a value below that observed in adults (75%).

In addition, the Pirouette program was used to perform the Princi

In addition, the Pirouette program was used to perform the Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA). Pembrolizumab clinical trial The chemometric

methods are particularly appropriate to provide insight into the Structure–Activity Relationships (SAR) when one is dealing with systems depending on many variables (Beebe and Pell, 1988). (PCA) and (HCA) are statistical methods used in the recognition of standards in multivaried studies (Da Silva et al., 2004, Weber et al., 2005 and Calgarotto et al., 2007). The properties calculated by DFT were auto-scaled using the Fisher weight (Costa and Takahata, 2003). The differences in the calculated properties are able to better discriminate the relationship between the structures of the sesquiterpene lactone derivative compounds and their biological activities. Results are presented

as the mean values ± S.D., obtained from the indicated number of tested animals. The statistical significance of differences between groups was evaluated using Student’s unpaired t-test. A P-value < 0.05 was considered to indicate significance. Myonecrosis, muscle tissue damage, is a common consequence of envenoming by snakes of the Bothrops genus and Selleckchem Raf inhibitor this effect is, partially, caused by PLA2 (Gutiérrez, 2002 and Soares et al., 2004). Compounds Lac01 and Lac02 reduced myotoxicity by approximately 70%, when compared to the PLA2 control assay (Fig. 2). Compounds Lac03 and Lac04 reduced the myotoxic activity by approximately 56%, while compounds Lac05–Lac08 did not demonstrate any activity against myotoxic effects. Edema-inducing activity is a pharmacological activity that depends upon the combined action

of various toxins, including PLA2 (Soares and Giglio, 2003 and Soares et al., 2004). Fig. 3 shows that, after a 2 h period, Lac01–Lac04 reduces the levels of edema-inducing activity of PLA2 to 40–50%, Anacetrapib when compared to the PLA2 control experiment. In this same period, the compounds Lac05–Lac08 reduced edema levels to only 90%. The action of PLA2 from B. jararacussu on the micellar substrate, HPGP, reflects the classical behavior of a Michaelian enzyme, not only in the presence of small concentrations of the lactone compounds (1–4 μM), but also in their absence (graphics not show) ( Souza et al., 2008 and Da Silva et al., 2008a). The kinetic parameters obtained in this study are shown in Table 1 and Fig. 4. Fig. 4 demonstrates that, in all the tests, the maximum velocity of the enzyme (Vmax) varied in function of the presence of growing concentrations of inhibitor compounds. Lac01 and Lac02 were the more efficient inhibitors and reduce the enzymatic activity around 80–90% ( Fig. 4A).

The LAP ELISA measured Latent TGF-β1 without preceding acidificat

The LAP ELISA measured Latent TGF-β1 without preceding acidification of human samples and did not cross-react with LAP2 or − 3. No cross-reactivity of the LAP ELISA with Latent TGF-β in bovine serum was found making the ELISA suitable also for human cell supernatants containing bovine serum. BALB/c mice were immunized subcutaneously with 10 μg recombinant human (rh) Latent TGF-β1 (R&D Systems, Abingdon, UK) in 50 μl phosphate-buffered saline (PBS) and 50 μl Freund’s complete

adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Mice were boosted using Freund’s incomplete adjuvant week 4 and 7 (Sigma). At week 27, 10 μg antigen in PBS was given intraperitoneally and 3 days later spleen cells were fused with Sp2/0 cells as described (elGhazali et al., 1993). Positive hybridomas Ibrutinib identified by indirect ELISA were subcloned E7080 mouse and cultured. MAbs were purified and biotinylated as described (Zuber et al., 2005). Mice were housed and handled at the Karolinska Institute, Solna, Sweden, according to the guidelines of the Swedish Ethical Committee for Animal Protection. Maxisorp 96-well plates (Nunc, Roskilde, Denmark) were

coated for 16 h at 4 °C with 2 μg/ml of rh LAP1, Latent TGF-β1 or TGF-β1 (R&D Systems) in 100 μl PBS. Other assay steps were at room temperature (RT), using 100 μl/well. Five washes using PBS with 0.1% Tween 20 were made between assay steps. After coating, wells were blocked for 1 h with incubation for buffer (PBS with 0.05% Tween 20 and 0.1% bovine serum albumin). Hybridoma supernatants were diluted in incubation buffer and incubated 2 h. Next, goat-anti-mouse IgG alkaline phosphatase conjugate (Mabtech, Nacka Strand, Sweden) was added and incubated for 1 h, followed by development with para-nitrophenyl phosphate (Sigma) and absorbance measurement (405 nm) by an ELISA reader (Labsystems, Helsinki, Finland). In the LAP ELISA, mAb MT593 (IgG1) was coated at 2 μg/ml and wells were blocked as above. Volumes and temperature for all assay steps and washes in between

were as above. Human plasma was diluted in an ELISA diluent (Mabtech) preventing potential interference by heterophilic antibodies in the samples (Bolstad et al., 2011). Also rh LAP1, Latent TGF-β1 and TGF-β1 and other samples were diluted in ELISA diluent. ELISA diluent was added to acidified samples after the acid treatment. After sample incubation for 2 h, mAb MT517-biotin (IgG1) at 1 μg/ml in ELISA diluent was incubated 1 h followed by streptavidin-horse radish peroxidase conjugate (SA-HRP; Mabtech) in incubation buffer for 1 h. The assay was developed with 3,3′,5,5′-tetramethylbenzidine substrate (Mabtech) and stopped with 1 M H2SO4 followed by absorbance measurement (450 nm). The same protocol was used for TGF-β1 ELISA (Human TGF-β1 DuoSet kit specific for TGF-β1; R&D Systems). Sample levels were determined against standards using rhLAP1 in the LAP ELISA and rhTGF-β1 in the TGF-β1 ELISA.

5% According to the National Cancer Institute, Surveillance, Epi

5%. According to the National Cancer Institute, Surveillance, Epidemiology, and End Results Program (Bethesda, MD) database, the 2-year survival rate was only 22% to 42% in patients with

stage III gastric cancer [18], signaling pathway which appears to be shorter than the results of the ACTS-GC and the CLASSIC trials. However, almost 70% of patients enrolled in this study had AJCC stage III disease, which was more advanced than the characteristics of those two trials. This difference may influence survival times. One controversial issue in the surgical management of gastric cancer is the optimal extent of lymph node dissection. Some large prospective clinical studies in western countries have shown that there was no difference in the 5-year survival rate among patients who underwent D1 versus D2 resection and that mortality related to surgery was higher in the D2 group [19], [20] and [21]. However, in Japan and other Asian countries, clinical studies have shown that the D2 operation can reduce postoperative local recurrence rates compared with D1 resection and that complication and operative mortality rates are very low [22]. In this study, 22 patients (68.8%) received a D2 resection, and 10 patients (31.3%) underwent a D1 operation. The median DFS of these two groups was significantly different (15 months for D1 and

18 months for D2 dissections; P = .043), suggesting that D2 lymphadenectomy may provide a survival benefit. Enzalutamide datasheet This result may also suggest that patients who undergo a radical

D2 dissection may also benefit more from adjuvant DCF chemotherapy. For patients who have had a D1 resection, DCF adjuvant chemotherapy may not be effective enough. The addition Nintedanib (BIBF 1120) of radiotherapy in these subgroups may be of paramount importance on the basis of the results of the US Intergroup trial INT0116 [23] and two recently published meta-analyses [24] and [25]. This study showed that neutropenia and febrile neutropenia occurred most frequently in patients treated with adjuvant DCF chemotherapy. The incidence of grade 3/4 neutropenia was high (at 56.4%), and febrile neutropenia occurred in 12.5% of patients. Other grade 3/4 adverse events developed in less than 10% of patients. There were no chemotherapy-related deaths in our study. The adverse events were manageable, and nonhematologic AE were more tolerable than in some previous studies. In TAX-325, the rates of any grade 3 or 4 toxicity during therapy were high when triple therapy was used (81%), and the most frequent grade 3/4 adverse events were neutropenia (30%) and diarrhea (20%) [17]. In summary, our results support the use of combination chemotherapy with DCF as a new approach to adjuvant treatment in patients with gastric cancer who have undergone radical surgery. We suggest further investigation of this adjuvant regimen. The authors report no conflicts of interest. The authors thank all the patients who participated in this study.

“Toxins from animal venoms with cytolytic activity play an

“Toxins from animal venoms with cytolytic activity play an important role in offensive and defensive actions in different organisms. In general, these roles are achieved by enzymatic cell lysis by phospholipases A2 and C.

However, a wide variety of cytolytic proteins and peptides lacking enzymatic activity have been isolated from reptilian, amphibian, insect, cnidaria, microbial and mammalian origins (Bernheimer and Rudy, 1986, Brinkman and Burnell, 2008, Frazão et al., 2012 and Kini and Evans, 1989). Differently from phospholipases, whose hemolytic activity is due to their ability to destroy cell membranes, most of those non-enzymatic proteins and peptides lyses cells by forming discrete transmembrane pores. Small osmoticants Trametinib can move in or out of the cell through those pores, while larger molecules such as proteins cannot. Thus the cell interior becomes hyperosmotic, attracting a net influx of water, which results in a sustained cell swelling and may

result in subsequent lysis (Menestrina et al., 1994). Pore-forming toxins interact to either lipids or proteins in the external cell membrane. It has been demonstrated that some toxins interact with erythrocyte membrane glycoproteins, such as glycophorin or band 3 (Garland and Buckley, 1988). Cytolytic activity on erythrocytes has been described for CDK inhibitor numerous animal venoms, including fish venoms, which exhibit high in vitro species-specific hemolytic activity. Hemolytic effect has been demonstrated in Pterois volitans, Pterois antennata ( Kiriake and

Shiomi, 2011), Scorpaena guttata ( Carlson et al., 1971), Scorpaena plumieri ( Andrich et al., 2010 and Carrijo et al., 2005), Synanceja verrucosa ( Garnier et al., 1995), Thalassophryne natterei ( Lopes-Ferreira et al., 1998 and Lopes-Ferreira et al., 2001) and Trachinus draco fish venoms ( Chhatwal and Dreyer, 1992). The hemolytic action of these venoms is very specific for rabbit erythrocytes. Erythrocytes from human, pig and chicken are resistant to hemolysis and weak hemolytic activity Tangeritin is observed on mice and cattle erythrocytes ( Chhatwal and Dreyer, 1992 and Kreger, 1991). Because fish venoms lack phospholipase A2 activity, this hemolytic action on erythrocytes can be seen as a direct hemolysis ( Khoo et al., 1992). Chhatwal and Dreyer (1992) suggested that the hemolytic activity of the T. draco venom is preceded by the binding of the hemolytic component to a protein receptor on the surface of erythrocytes. Recently, a new cytolytic toxin, referred to as Sp-CTx has been purified from the venom of the scorpionfish S. plumieri by our group ( Andrich et al., 2010).

These venom components can act on the nervous, cardiovascular, an

These venom components can act on the nervous, cardiovascular, and immunological systems of mammalians. Some inflammatory, vasoactive and thrombogenic substances, such as serotonin, histamine, leukotrienes, dopamine, thromboxanes and bradykinin, have been found in wasp venoms (Levine, 1976). The present study describes for the first time the lethality of S. cyanea venom on mice and some pharmacological activities induced by this venom in some cells or tissues. S. cyanea is widely distributed in Brazil and their nests are commonly

found in tree trunks located in urban areas ( Elisei et al., 2005 and Andena et al., 2009). The LD50 of S. cyanea is 16.68 mg/kg of mice. Lethality assays on mice showed LD50 of 2.4 mg/kg for Polistes canadense venom ( Schmidt, 1990) and 3.5 mg/kg for Vespula squamosa venom ( Schmidt et al., 1980). S. cyanea venom is 6.9 and 4.7 times less toxic

than Ku-0059436 manufacturer P. canadense and V. squamosa venom, respectively. Thus, although it has been shown that the S. cyanea venom is less toxic than the other wasp venoms that had their lethality tested so far, it is important to note that S. cyanea is a very aggressive social wasp and, for this reason, the seriousness of accidents involving humans cannot be discounted. The most prominent acute symptoms observed in accidents involving Bak apoptosis inoculation of wasp venom are the formation of a localized cutaneous oedema, pain and local lesions, these symptoms being found even in higher vertebrates, such as man (Griesbacher et al., 1998 and Mortari et al., 2005). Wasp venom-induced hindpaw oedema in Wistar rats after subplantar injection was observed in this study, at the minimum dose of 12.5 μg/paw, and also in a 48 h experiment with the African paper wasp P. fuscatus Ribonucleotide reductase venom, in which was found a conspicuous dose- and time-dependent oedema production; the lowest assayed dose being 20 μg/paw, sufficient to induce significant oedema ( Eno, 1997). In another study, it was also demonstrated that the venom of three different social wasps,

P. occidentalis, Polybia ignobilis and P. paulista, produced oedema after subplantar injection, and the minimum active dose was 10 μg/rat paw ( Mortari et al., 2005). Yshii et al. (2009) also observed paw oedema induction by Polistes lanio lanio paper wasp venom (7 μg/mouse paw) during a four-hour experiment and this effect was time-dependent. These differences observed in paw oedema induction by distinct wasp venoms can be due to variabilities in venom composition. Histamine and/or serotonin in venom are often related to the immediate local hindpaw oedema observed following venom injection from wasps such as P. fuscatus ( Eno, 1997), Vespula vulgaris ( Griesbacher et al., 1998), Vespa basalis ( Ho and Hwang, 1991) and P. lanio lanio ( Yshii et al., 2009).

During granules secretion, phagocytosis and killing of pathogens,

During granules secretion, phagocytosis and killing of pathogens, levels of calcium in the cytosol are usually increased ( Lee et al., 2003). In cells co-treated with MGO/high glucose (GM group) there was an increase in MPO enzyme activity and consequently in the production of hypochlorous acid (Table 1),

whereas neither high glucose nor MGO alone yielded the same effect (data not shown). Myeloperoxidase is an enzyme stored in azurophilic granules of polymorphonuclear neutrophils and released into extracellular fluid during inflammatory processes. Several studies have shown its involvement in oxidative stress and inflammation. Recently, MPO has been considered as a possible marker this website of plaque instability and a useful tool for the prognostic evaluation of patients with Venetoclax solubility dmso coronary artery disease (Gustapane et al., 2011). Possibly, increased release and activity of MPO in neutrophils promoted by co-treatment of neutrophils with MGO/high glucose could contribute to the development of the micro- and macro-vascular complications observed in diabetic condition. In contrast, cells treated with antioxidants promoted a marked reduction in the MPO and HClO production along with a drastic reduction in all reactive oxygen species. This effect was not observed when cells were treated with either astaxanthin or vitamin C alone. Superoxide

is a physiological substrate for MPO and their interactions are central to an important host defense mechanism. When released by neutrophils, MPO enzyme operates in the presence of a flux of superoxide. Winterbourn and Kettle (2004) showed that superoxide has a profound influence on oxidative reactions catalysed by MPO. It reacts directly with the enzyme to modulate production of hypochlorous acid. Within neutrophil phagosomes, where MPO acts by killing micro-organisms, it

may be the preferred substrate for the enzyme. Superoxide also reacts rapidly with radicals generated by MPO forming different species. These species are likely to be toxic and contribute to the pathophysiological actions of MPO (Winterbourn and Kettle, 2004). Therefore, reduced superoxide anion and hydrogen ID-8 peroxide production promoted by astaxanthin and vitamin C can be involved in the reduced MPO and HClO production as well as a direct scavenger effect promoted by antioxidants. In addition, the phagocytic capacity of neutrophils and G6PDH activity, key enzyme of pentose pathway involved in NADPH formation, were decreased in cells after treatment with MGO/high glucose. A decrease in the phagocytic capacity accompanied by a decrease in NADPH availability could mean minor neutrophil effectiveness to destroy pathogens. This fact has been associated with the impairment in neutrophil function observed in diabetes (Lecube et al., 2011). Decreased phagocytic capacity by induced by MGO/high glucose was prevented by treatment of cells with the combination of antioxidants astaxanthin and vitamin C.

56 CA titers typically range between 512 and 32000 DAT is always

56 CA titers typically range between 512 and 32000. DAT is always positive for C3d.[3] and [56] Most

reported patients have been adults, and AIHA typically occurs during the second or third week after the febrile illness has started.56 In most published cases the PD0325901 ic50 onset has been sudden with pallor, jaundice and, sometimes, prostration. Intravascular hemolysis, as evidenced by hemoglobinuria, has been reported in several patients. In general, the prognosis is good and the hemolytic complication is self-remitting within 4–6 weeks, although a lethal course has been described in one patient.[55] and [56] A number of case reports have been published on CA mediated AIHA in infectious mononucleosis with confirmed Epstein–Barr virus (EBV) etiology.[57], [58], [59] and [60] As compared to M. pneumoniae pneumonia, however, infectious mononucleosis is an infrequent cause of AIHA, accounting for approximately 1% of the cases. [1] and [2] Conversely, the frequency of clinically significant hemolysis in EBV infections is unknown but probably low. Hospital-based data have indicated check details that hematological complications, being generally mild and including several manifestations

other than AIHA, occur in 25–50% of patients with EBV infection. 59 Since patient selection will influence such figures and most individuals with infectious mononucleosis are not hospitalized, the frequency is probably much lower among patients with EBV-infection in the community. CA found in EBV-infections are polyclonal and almost invariably specific for the i-antigen.[57], [60] and [61] The immunoglobulin class may be either IgM or IgG.60 Rheumatoid factor-like IgM-IgG complexes have also been reported to act as CA in single cases.60 In most published reports, DAT has been positive for C3d only. Anti-i titers are usually modest, typically at 256–512, and the hemolytic anemia is transient and generally mild.60 Several authors have reported on transient CAS mediated

by anti-i autoantibodies following cytomegalovirus (CMV) infection.[60] and [61] Rarely, CA-mediated hemolysis has been described in adenovirus infections, influenza A, varicella, rubella, Legionella pneumophilica pneumonia, Celecoxib listeriosis and pneumonia caused by Chlamydia species. [11], [60], [62], [63], [64] and [65] We observed a slight, transient CAS in an otherwise healthy 23 year old man two weeks after a Chlamydia pneumoniae pneumonia. Severe CAS with a prolonged course and cryoglobulin activity of the CA has been reported following Escherichia coli lung infection. 22 Autoantibody specificities in these rare cases have included anti-I, anti-i and anti-Pr. Cold-antibody AIHA with infectious etiology typically involves a young adult or adolescent with M. pneumoniae pneumonia or infectious mononucleosis. Anemia or hemoglobinuria in such patients should immediately raise the suspicion of secondary AIHA.

MBD2 mediates silencing by recruiting the NuRD complex to methyla

MBD2 mediates silencing by recruiting the NuRD complex to methylated DNA.62 and 63 Structural studies of the MBD2-NuRD complex have identified a critical coiled-coil protein interaction between MBD2 and p66α/β, another NuRD complex component. Enforced expression of the p66 coiled-coil protein results in release of the Mi2β chromatin remodeling ATPase from the NuRD complex, and derepression of the silenced embryonic and fetal β-type globin genes, presumably by decoupling MBD2 from the NuRD chromatin remodeling function.60 A closely related member of the MBD family,

MBD3, also associates with a NuRD complex, but does not bind to methylated vs nonmethylated DNA with high affinity.58 and 64 Moreover, the presence of MBD2 and MBD3 in association with NuRD complex appears to be mutually exclusive.65 MBD3-NuRD Ibrutinib purchase is associated with the ɣ-globin gene promoter primarily through association with the GATA1 transcription factor–associated Protein Tyrosine Kinase inhibitor protein, friend of GATA1 (FOG1),32 and 33 or other complexes.66 Disruption of expression of the Mi2β subunit of NuRD results in increased ɣ-globin gene expression in transgenic mice,34 cultured mouse chemical inducer of dimerization (CID) hematopoietic cells

bearing a human β-globin gene locus, and cultured primary human erythroid cells.67 Recently, it was shown that as little as a 50% knockdown of Mi2β in primary human erythroid cells results in a ∼10-fold increase in ɣ-globin gene expression without affecting erythroid differentiation, compared with control CD34+ progenitor–derived erythroid cells treated with scramble short hairpin RNA.67 The degree of differentiation in control cells in these studies leads to a level of 1% ɣ/ɣ+β RNA, which is comparable with normal adult reticulocyte

levels. Interestingly, in these studies, the effect of Mi2β on ɣ-globin gene silencing did not appear to be chiefly because of an effect on MBD2-NuRD for or MBD3-NuRD. Rather at least part of the effect was through downregulation of BCL11A and KLF1 in Mi2β knockdown erythroid cells. The purposed relationships of MBD2-NuRD, MBD3-NuRD, and Mi2β in ɣ-globin gene silencing in the context of other major epigenetic regulatory factors are depicted in Fig 1. 67 On the basis of the preponderance of evidence, it appears that MBD2 plays a greater role than MBD3 in silencing ɣ-globin gene expression, whereas Mi2β plays a greater role than either MBD2 or MBD3. Increased histone acetylation has long been posited to be associated with decompressed chromatin and active gene expression.68 and 69 The writers for histone acetylation are histone acetyltransferases including P300/CBP (CRE3 binding protein), PCAF, and TAF(11)250 (TBP associated factor),70 as well as histone deacetylases (HDACs, which might be more properly thought of as “erasers”). The complexity of histone acetylation and its relationship to gene regulation have been intensively studied and will not be reviewed in detail here.

2, Fig  3 and Fig  4) Alexafluor 488 labeled BSA as the control

2, Fig. 3 and Fig. 4). Alexafluor 488 labeled BSA as the control culture did not bind to any of these cell lines (data not shown). Our binding data for pure BoNT/A confirmed previously published research in which the purified BoNT/A bound to cell lines of neuronal origin, but not to those of non-neuronal origin (Kurokawa et al., 1987). But it has not been reported before that in addition binding to human neuronal cells, both Transmembrane Transporters inhibitor BoNT/A complex and NAPs can also bind to non-neuronal cells such as lymphoblasts, skeletal muscle cells, and fibroblasts. Although BoNT/A in its purified and complex forms all bind to

SH-SY5Y, the intracellular responses of the SH-SY5Y cells to these BoNT/A components have not been well studied. Among all the 28 human inflammatory cytokines tested, there were three categories of cytokine release responses: (1) no detectable release, (2) release but no significant differences between BoNT/A, BoNT/A complex or NAPs treatment, and (3) significantly different release induced by BoNT/A, BoNT/A complex or NAPs. The release of the following thirteen learn more cytokines was below the limit of detection after exposure to different components of BoNT/A associated proteins: IL-1β, MIG, IL-1ra, IL-2, IL-5, IL-17, Eotaxin, basic FGF, G-CSF, GM-CSF, MIP-1α, MIP-1β, and PDFF-BB (Supplementary Table

S1). For the following seven cytokines positive releases were detected, but there were no significant changes after the treatment with BoNT/A, BoNT/A complex, Silibinin or NAPs: IL-4, IL-7, IL-9, IL-10, IL-12, IL-13, and IFN-γ (Τable S1). The cytokines

which were significantly induced by different components of BoNT/A and its associated proteins are listed in Table 1. Pure 150 kDa BoNT/A did not significantly increase the release of any inflammatory cytokines from SH-SY5Y cells, compared to BSA control. Exposure to NAPs or BoNT/A complex, however, increased the release of multiple inflammatory cytokines. The release of IL-6, MCP-1, and VEGF were all significantly increased after exposure to BoNT/A complex and NAPs compared with control. In addition, BoNT/A complex induced a significant increase of MCP-1 release compared with NAPs. BoNT/A complex, but not NAPs or BoNT/A, also induced dramatic increase in IP-10, IL-8, TNF-α, and RANTES compared with the control. These results suggest the possibility of NAPs may contribute to local and systemic inflammatory process after the administration of NAPs-containing BoNT/A drugs in patients. Over five million patients are being treated with botulinum neurotoxins globally (Singh et al., 2010), and because of the safety concerns of this being the most toxic substance known to mankind, the United States Food and Drug Administration (US FDA) has designated all botulinum neurotoxin based drugs for black box label (Kuehn, 2009). There have been reports of side effects such as cognition issues and flu-like symptoms from BoNT-based therapeutics (Alam et al., 2002, Costa et al., 2005 and Cote et al.